CA2227895C - Multiplexed analysis of clinical specimens apparatus and methods - Google Patents

Multiplexed analysis of clinical specimens apparatus and methods Download PDF

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Publication number
CA2227895C
CA2227895C CA2227895A CA2227895A CA2227895C CA 2227895 C CA2227895 C CA 2227895C CA 2227895 A CA2227895 A CA 2227895A CA 2227895 A CA2227895 A CA 2227895A CA 2227895 C CA2227895 C CA 2227895C
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bead
beads
subset
interest
analyte
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CA2227895A1 (en
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Van S. Chandler
R. Jerrold Fulton
Mark B. Chandler
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Luminex Corp
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Luminex Corp
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Priority claimed from US08/542,401 external-priority patent/US5736330A/en
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/1012Calibrating particle analysers; References therefor
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • G01N15/1456Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5094Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
    • G01N15/149
    • G01N2015/1014
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • G01N2015/1477Multiparameters
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • G01N2015/1486Counting the particles
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • G01N2015/1488Methods for deciding
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/973Simultaneous determination of more than one analyte

Abstract

A method for the multiplexed diagnostic and genetic analysis of enzymes, DNA
fragments, antibodies, and other biomolecules comprises the steps of constructing an appropriately labeled beadset, exposing the beadset to a clinical sample, and analyzing the combined sample/beadset by flow cytometry is disclosed. Flow cytometric measurements are used to classify, in real-time, beads within an exposed beadset and textual explanations, based on the accumulated data obtained during real-time analysis, are generated for the user. The inventive technology enables the simultaneous, and automated, detection and interpretation of multiple biomolecules or DNA sequences in real-time while also reducing the cost of performing diagnostic and genetic assays.

Description

MULTIPLEXED ANALYSIS OF CLINICAL
SPECIMENS APPARATUS AND METHODS
Microfiche appendix A contains a listing of selected Visual Basic and C
programming source code in accordance with the inventive multiplexed assay method.
Microfiche appendix A, comprising I sheet having a total of 58 frames, contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by io anyone of the patent disclosure, as it appears in the Patent and Trademark Office patent files or records, but otherwise reserves all copyright rights whatsoever.

The invention relates generally to laboratory diagnostic and genetic analysis and, more particularly, to a flow cytometric method for the simultaneous and multiplexed diagnostic and genetic analysis of clinical specimens.

Analysis of clinical specimens is important in science and medicine. A wide variety of assays to determine qualitative and/or quantitative characteristics of a specimen are known in the art. Detection of multiple analytes, or separately identifiable characteristics of one or more analytes, through single-step assay processes are presently not possible or, to the extent possible, have provided only very limited capability and have not yielded satisfactory results. Some of the reasons for these disappointing results include the extended times typically required to enable the detection and classification of multiple analytes, the inherent limitations of known reagents, the low sensitivities achievable in prior art assays which often lead to significant analytical errors and the unwieldy collection, classification, and analysis of prior art algorithms vis a. vis the large amounts of data obtained and the subsequent computational requirements to analyze that data.

Clearly, it would be an improvement in the art to have adequate apparatus and methods for reliably performing real-time multiple determinations, substantially simultaneously, through a single or limited step assay process. A capability to perform simultaneous, multiple SUBSTITUTE SHEET (RULE 26) determinations in a single assay process is known as "multiplexing" and a process to implement such a capability is a "multiplexed assay."
Flow Cytometry One well known prior art technique used in assay procedures for which a multiplexed assay capability would be particularly advantageous is flow cytometry. Flow cytometry is an optical technique that analyzes particular particles in a fluid mixture based on the particles' optical characteristics using an instrument known as a flow cytometer.
'Background information on flow cytometry may be found in Shapiro, "Practical Flow Cytometry," Third Ed. (Alan R.
Liss, Inc. 1995); and Melamed et al., "Flow Cytometry and Sorting," Second Ed.
(Wiley-Liss to 1990), which may be referred to for further details.

Flow cytometers hydrodynamically focus a fluid suspension, of particles into a thin stream so that the particles flow down the stream in substantially single file and pass through an examination zone. A focused light beam, such as a laser beam illuminates the particles as they flow through the examination zone. Optical detectors within the flow cytometer measure certain characteristics of the light as it interacts with the particles. Commonly used flow cytometers such as the Becton-Dickinson Immunocytometry Systems "FACSCAN" (San Jose, CA) can measure forward light scatter (generally correlated with the refractive index and size of the particle being illuminated), side light scatter (generally correlated with the particle's size), and particle fluorescence at. one or more wavelengths. (Fluorescence is typically imparted by incorporating, or attaching a fluorochrome within the particle.) Flow cytometers and various techniques for their use are described in, generally, in "Practical Flow Cytometry" by Howard M. Shapiro (Alan R. Liss, Inc., 1985) and "Flow Cytometry and Sorting, Second Edition" edited by Melamed et al. (Wiley-Liss, 1990).

One skilled in the art will recognize that one type of "particle" analyzed by a flow cytometer may be man-made microspheres or beads. Microspheres or beads for use in flow cytometry are generally known in the art and may be obtained from manufacturers such as Spherotech (Libertyville, IL), and Molecular Probes (Eugene, OR).
Although a multiplexed analysis capability theoretically would provide enormous = benefits in the art of flow cytometry, very little multiplexing capability has been previously achieved. Prior multiplexed assays have obtained only a limited number of determinations. A
review of some of these prior art techniques is provided by McHugh, "Flow Microsphere Immunoassay for the Quantitative and Simultaneous Detection of Multiple Soluble Analytes," in Methods in Cell Biology, 42, Part B, (Academic Press, 1994). For example, McHugh et al., "Microsphere-Based Fluorescence Immunoassays Using Flow Cytometry Instrumentation," in Clinical Flow Cytometry Ed. K.D. Bauer, et al., Williams and Williams, Baltimore, MD, 1993, 535-544, describe an assay where microspheres of different sizes are used as supports and the 1o identification of microspheres associated with different analytes was based on distinguishing a microsphere's size. Other references in this area include Lindmo, et al., "Immunometric Assay by Flow Cytometry Using Mixtures of Two Particle Types of Different Affinity,"
J. Immun.
Meth., 126, 183-189 (1990); McHugh, "Flow Cytometry and the Application of Microsphere-Based Fluorescence Immunoassays," Immunochemica, 5, 116 (1991); Horan et al., "Fluid Phase Particle Fluorescence Analysis: Rheumatoid Factor Specificity Evaluated by Laser Flow Cytophotometry" in Immunoassays in the Clinical Laboratory, 185-198 (Liss 1979); Wilson et al., "A New Microsphere-Based Immunofluorescence Assay Using Flow Cytometry,"
J. Immunological Methods, 107, 225-230 (1988); and Fulwyler et al., "Flow Microsphere Immunoassay for the Quantitative and Simultaneous Detection of Multiple Soluble Analytes,"
Meth. Cell Biol., 33, 613-629 (1990).

The above cited methods have been unsatisfactory as applied to provide a fully multiplexed assay capable of real-time analysis of more than a few different analytes. For example, certain of the assay methods replaced a single ELISA procedure with a flow cytometer-based assay. These methods were based on only a few characteristics of the particles under analysis and enabled simultaneous determination of only a very few analytes in the assay.
Also, the analytic determinations made were hampered due to software limitations including the inability to perform real-time processing of the acquired assay data. In summary, although it has been previously hypothesized that flow cytometry may possibly be adapted to operate and provide benefit in a multiple analyte assay process, such an adaptation has not in reality been accomplished.

Analysis of Genetic Information The availability of genetic information and association of disease with mutation(s) of critical genes has generated a rich field of clinical analysis. In particular, the use polymerase chain reaction (PCR) and its variants have facilitated genetic analysis. A
major advance in this field describes a powerful flow cytometric assay for PCR products, which may be multiplexed in accordance with the present invention. A multiplexed flow cylometric to assay for PCR reaction products would provide a significant advantage in the field of genetic analysis.

Recent advances in genetic analyses have provided a wealth of information regarding specific mutations occurring in particular genes in given disease states.
Consequently, use of an individual's genetic information in diagnosis of disease is becoming increasingly prevalent. Genes responsible for disease have been cloned and characterized in a number of cases, and it has been shown that responsible genetic defects may be a gross gene alteration, a small gene alteration, or even in some cases, a point mutation. There are a number of reported examples of diseases caused by genetic mutations. Testing of gene expression by analysis of cDNA or mRNA, and testing of normal genes and alleles, as in cases of tissue typing and forensics, are becoming widespread.
Other uses of DNA analysis, for example in paternity testing, etc., are also important and can be used in accordance with the invention.

Current techniques for genetic analysis have been greatly facilitated by the development and use of polymerase chain reaction (PCR) to amplify selected segments of DNA. The power and sensitivity of the PCR has prompted. its application to a wide variety of analytical problems in which detection of DNA or RNA sequences is required.
PCR is capable of amplifying short fragments of DNA, providing short (20 bases or more) = nucleotides are supplied as primers. The primers anneal to either end of a span of denatured DNA
target and, upon renaturation, enzymes synthesize the intervening complementary sequences by extending the primer along the target strand. During denaturation, the temperature is raised to break apart the target and newly synthesized complementary sequence. Upon cooling, renaturation and annealing, primers bind to the target and the newly made opposite strand and now the primer is extended again creating the complement. The result is that in each cycle of heating and renaturation followed by primer extension, the amount of target sequence is doubled.

One major difficulty with adoption of PCR is the cumbersome nature of the methods of analyzing the reaction's amplified DNA products. Methods for detecting genetic abnormalities and PCR products have been described but they are cumbersome and time consuming.
For example, U.S. Patent No. 5,429,923 issued July 4, 1995 to Seidman, et al., describes a method for detecting mutations in persons having, or suspected of having, hypertrophic cardiomyopathy. That method 1s involves amplifying a DNA sequence suspected of containing the disease associated mutation, combining the amplified product with an RNA probe to produce an RNA-DNA hybrid and detecting the mutation by digesting unhybridized portions of the RNA strand by treating the hybridized product with an RNAse to detect mutations, and then measuring the size of the products of the RNAse reaction to determine whether cleavage of the RNA molecule has occurred.
Other methods used for detecting mutations in DNA sequences, including direct sequencing methods (Maxim and Gilbert, Proc. Natl. Acad. Sci. U.S.A., 74, 560-564, 1977); PCR
amplification of specific alleles, PASA (Botttema and Sommer, Muta. Res., 288, 93-102, 1993);
and reverse dot blot method (Kawasaki, et al., Methods in Enzymology, 218, 369-81, 1993) have been described. These techniques, while useful, are time consuming and cumbersome and for that reason are not readily adaptable to diagnostic assays for use on a large scale.

At least one use of flow cytometry for the assay of a PCR product has been reported but that assay has not been adapted to multiplexing. See Vlieger et al., "Quantitation of Polymerase Chain Reaction Products by Hybridization-Based Assays with Fluorescent Colorimetric, or WO 97/14028 PCT/US96/16198.
Chemiluminescent Detection," Anal. Biochem., 205, 1-7 (1992). In Vlieger et al. a PCR
product was labeled using primers that contained biotinylated nucleotides.
Unreacted primers were first removed and the amplified portion annealed with a labeled complementary probe in solution. Beaded microspheres of avidin were then attached to the annealed complementary s material. The avidin beads bearing the annealed complementary material were then processed by a flow cytometer. The procedure was limited, inter alia, in that avidin beads having only a single specificity were employed. Further, real-time analysis of the assay's data was not possible.

io Data Manipulation The large volume of data typically generated during flow cytometric multiple analyte assays, combined with the limited capabilities of prior techniques to collect, sort and analyze such data have provided significant obstacles in achieving a satisfactory multiplexed assay. The computing methods used in prior art flow cytometric analyses have generally been insufficient 15 and unsuited for accurately and timely analyzing large volumes of data such as would be generated by multiplexed assays; particularly when more than two analytes (or properties of a single analyte) are to be simultaneously determined.

The present invention enables the simultaneous determination of multiple distinct 20 analytes to a far greater degree than existing techniques. Further, the invention provides an improved data classification and analysis methodology that enables the meaningful analysis of highly multiplexed assays in real-time. The invention is broadly applicable to multiplexed analysis of a number of analytes in a host of bioassays in which there is currently a need in the art.
The present invention provides improved methods, instrumentation, and products for detecting multiple analytes in a fluid sample by flow cytometric analysis and for analyzing and presenting the data in real-time. An advantage of the invention is that it allows one rapidly and simultaneously to detect a wide variety of analytes of interest in a single assay step.

WO 97/14028 PCT/US961'16198 The invention employs a pool of bead subsets. The individual subsets are prepared so that beads within a subset are relatively homogeneous but differ in at least one distinguishing characteristic from beads in any other subset. Therefore, the subset to which a bead belongs can readily be determined after beads from different subsets are pooled.
In a preferred embodiment, the beads within each subset are uniform with respect to at least three and preferably four known classification parameter values measured with a flow cytometer: e.g., forward light scatter (C1) which generally correlates with size and refractive index; side light scatter (C2) which generally correlates with size; and fluorescent emission in at least one wavelength (C3), and preferably in two wavelengths (C3 and C4), which generally io results from the presence of fluorochrome(s) in or on the beads. Because beads from different subsets differ in at least one of the above listed classification parameters, and the classification parameters for each subset are known, a bead's subset identity can be verified during flow cytometric analysis of the pool in a single assay step and in real-time.

1s Prior to pooling subsets of beads to form a beadset, the beads within each subset can be coupled to a reactant that will specifically react with a given analyte of interest in a fluid sample to be tested. Usually, different subsets will be coupled to different reactants so as to detect different analytes. For example, subset I may be labeled so as to detect analyte A (AnA); subset 2 may be labeled so as to detect analyte B (AnB); etc.
At some point prior to assay, the variously labeled subsets are pooled. The pooled beads, or beadset, are then mixed with a fluid sample to test for analytes reactive with the various reactants bound to the beads. The system is designed so that reactions between the reactants on the bead surfaces and the corresponding analytes in the fluid sample will cause changes in the intensity of at least one additional fluorescent signal (Fm) emitted from a fluorochrome that fluoresces at a wavelength distinct from the wavelengths of classification parameters C3 or C4.
The Fm signal serves as a "measurement signal," that is, it indicates the extent to which the reactant on a given bead has undergone a reaction with its corresponding analyte. The F. signal may result from the addition to the assay mixture of fluorescently labeled "secondary" reagent that binds to the bead surface at the site where a reactant-analyte reaction has occurred.
When the mixture (pooled beads and fluid sample) is run through a flow cytometer, each bead is individually examined. The classification parameters, e.g., C1, C2, C3, and C4, are measured and used to classify each bead into the subset to which it belongs and, therefore, identify the analyte that the bead is designed to detect. The Fm value of the bead is determined to indicate the concentration of analyte of interest in the fluid sample. Not only are many beads from each subset rapidly evaluated in a single run, multiple subsets are evaluated in a single run.
Thus, in a single-pass and in real-time a sample is evaluated for multiple analytes. Measured Fm values for all beads assayed and classified as belonging to a given subset may be averaged or io otherwise manipulated statistically to give a single meaningful data point, displayed in histogram format to provide information about the distribution of Fm values within the subset, or analyzed as a function of time to provide information about the rate of a reaction involving that analyte.

In a preferred embodiment, the beads will have two or more fluorochromes incorporated within or on them so that each of the beads in a given subset will possess at least four different classification parameters, e.g., C1, C2, C3, and C4. For example, the beads may be made to contain a red fluorochrome (C3), such as nile red, and bear an orange fluorochrome (C4), such as Cy3 or phycoerythrin. A third fluorochrome, such as fluorescein, may be used as a source of the C,, or Fm signal. As those of skill in the art will recognize, additional fluorochromes may be used to generate additional Cõ signals. That is, given suitable fluorochromes and equipment, those of skill in the art may use multiple fluorochromes to measure a variety of Cõ or Fm values, thus expanding the multiplexing power of the system even further.

In certain applications designed for more quantitative analysis of analyte concentrations or for kinetic studies, multiple subsets of beads may be coupled to the same reactant but at varying concentrations so as to produce subsets of beads varying in density of bound reactant rather than in the type of reactant. In such an embodiment, the reactant associated with classification parameter C4, for example, may be incorporated directly into the reactive reagent that is coupled to the beads, thereby allowing C4 conveniently to serve as an indicator of density = of reactant on the bead surface as well as an indicator of reactant identity.

To prepare subsets varying in reactant density one may, for example, select, isolate, or prepare a starting panel of different subsets of beads, each subset differing from the other subsets in one or more of C1, C2, or C3. Each of those subsets may be further subdivided into a number of aliquots. Beads in each aliquot may be coupled with a reactant of choice that has been fluorescently labeled with a fluorochrome associated with C4 (e.g., Analyte A
labeled with Cy3) under conditions such that the concentration or density of reactant bound to the beads of each io aliquot will differ from that of each other aliquot in the subset.
Alternatively, an entire subset may be treated with the C4 fluorochrome under conditions that produce a heterogeneous distribution of C4 reactant on beads within the subset. The subset may then be sorted with a cell sorter on the basis of the intensity of C4 to yield further subsets that differ from one another in C4 intensity.
is One limitation of the alternative embodiment of using C4 labeled reactant as a classification agent is that one must design the system so that the value of C4 as a classification parameter is not lost. Therefore, one must take care to assure that the C4 intensities of all subsets carrying reagent A differs from the C4 intensities of all subsets carrying reagents B, C, 20 and so forth. Otherwise, C4 would not be useful as a parameter to discriminate reactant A from reactant B, etc.

With either embodiment, the number of subsets that can be prepared and used in practice of the invention is theoretically quite high, but in practice will depend, inter alia, on the level of 25 homogeneity within a subset and the precision of the measurements that are obtained with a flow cytometer. The infra-subset heterogeneity for a given parameter, e.g., forward angle light scatter C1, correlates inversely with the number of different subsets for that parameter that can be discriminated by flow cytometric assay. It is therefore desirable to prepare subsets so that the coefficients of variation for the value of each classification parameter (Cl, C2, C3, and C4) to be 30 used in a given analysis is minimized. Doing this will maximize the number of subsets that can be discriminated by the flow cytometer. Bead subsets may be subjected to flow cytometric sorting or other procedures at various different points in preparation or maintenance of the bead subsets to increase homogeneity within the subset. Of course, with simple assays designed to detect only a few different analytes, more heterogeneity can be allowed within a subset without s compromising the reliability of the assay.
In an illustrative embodiment set forth here to explain one manner in which the invention can work in practice, the beads are used to test for a variety of antibodies in a fluid sample. A
panel of bead subsets having known varying Cj, C2, C3, and C4 values is first prepared or otherwise obtained. The beads within each subset are then coupled to a given antigen of i0 interest. Each subset receives a different antigen. The subsets are then pooled to form an assay beadset and may be stored for later use and/or sold as a commercial test kit.

In the assay procedure, the beads are mixed with the fluid to be analyzed for antibodies reactive with the variety of antigens carried on the beads under conditions that will permit is antigen-antibody interaction. The beads are labeled with a "secondary"
reagent that binds to antibodies bound to the antigens on the beads and that also bears the measurement fluorochrome associated with parameter F. (e.g., fluorescein). A fluoresceinated antibody specific for immunoglobulin may be used for this purpose. The beads are then run through a flow cytometer, and each bead is classified by its characteristic classification parameters as belonging 20 to subset-1, subset-2, etc. At the same time, the presence of antibodies specific for antigen A, B, etc., can be detected by measuring green fluorescence, Fm, of each bead. The classification parameters C1, C2, C3, and C4 allow one to determine the subset to which a bead belongs, which serves as an identifier for the antigen carried on the bead. The Fm value of the bead indicates the extent to which the antibody reactive with that antigen is present in the sample.

Although assays for antibodies were used above as an illustration, those of ordinary skill in the art will recognize that the invention is not so limited in scope, but is widely applicable to detecting any of a number of analytes in a sample of interest. For example, the methods described here may be used to detect enzymes or DNA or virtually any analyte detectable by virtue of a given physical or chemical reaction. A number of suitable assay procedures for detection and quantification of enzymes and DNA (particularly as the result of a PCR process) are described in more detail below.

The present invention also provides a significant advance in the art by providing a rapid and sensitive flow cytometric assay for analysis of genetic sequences that is widely applicable to detection of RNA, differing alleles, and any of a number of genetic abnormalities. In general, the methods of the present invention employ a competitive hybridization assay using DNA coupled microspheres and fluorescent DNA probes. Probes and microsphere-linkedoligonucleotides could also include RNA, PNA, and non-natural nucleotide analogs.
In practice of the invention, oligonucleotides from a region of a gene of interest, often a polymorphic allele or a region to which a disease associated mutation has been mapped, are synthesized and coupled to a microsphere (bead) by standard techniques such as by carbodiimide coupling. A fluorescent oligonucleotide, complementary to the oligonucleotideon the bead, is also is synthesized. To perform a test in accordance with the invention, DNA which is to be tested is purified and either assayed unamplified, or subjected to amplification by PCR, RT-PCR, or LCR
amplification using standard techniques and PCR initiation probes directed to amplify the particular region of DNA of interest. The PCR product is then incubated with the beads under conditions sufficient to allow hybridization between the amplified DNA and the oligonucleotides present on the beads. A fluorescent DNA probe that is complementary to the oligonucleotide coupled to the beads is also added under competitive hybridization conditions.
Aliquots of the beads so reacted are then run through a flow cytometer and the intensity of fluorescence on each bead is measured to detect the level of fluorescence which indicates the presence or absence of given sequences in the samples.

For example, when beads labeled with an oligonucleotide probe corresponding to a non-mutated (wild-type) DNA segment are hybridized with the PCR product from an individual who has a non-mutated wild-type DNA sequence in the genetic region of interest, the PCR product will effect a significant competitive displacement of fluorescent oligonucleotide probe from the beads 3o and, therefore, cause a measurable decrease in fluorescence of the beads, e.g., as compared to a control reaction that did not receive PCR reaction product. If, on the other hand, a PCR product from an individual having a mutation in the region of interest is incubated with the beads bearing the wild-type probe, a significantly lesser degree of displacement and resulting decrease in intensity of fluorescence on the beads will be observed because the mutated PCR product will be a s less effective competitor for binding to the oligonucleotide coupled to the bead than the perfectly complementary fluorescent wild-type probe. Alternatively, the beads may be coupled to an oligonucleotide corresponding to a mutation known to be associated with a particular disease and similar principles applied. In the multiplexed analysis of nucleic acid sequences, bead subsets are prepared with all known, or possible, variants of the sequence of interest and then mixed to form a io bead set. The reactivity of the test sample, e.g. PCR product, with the wild-type sequence and other variants can then be assayed simultaneously. The relative reactivity of the PCR product with subsets bearing the wild-type or variant sequences identifies the sequence of the PCR product. The matrix of information derived from this type of competitive hybridization in which the test sequence and the entire panel of probe sequences react simultaneously allows identification of the is PCR product as wild-type, known mutant, or unknown mutant. The invention thus provides one with the ability to measure any of a number of genetic variations including point mutations, insertions, deletions, inversions, and alleles in a simple, exquisitely sensitive, and efficient format.

Figure 1 is a block diagram of an illustrative hardware system for performing a multiplex 20 assay method in accordance with the invention.

Figure 2 is a block diagram of an illustrative software system for performing a multiplex assay method in accordance with the invention.

25 Figure 3 is a flow-chart for a preprocessing phase in accordance with the inventive multiplexed assay method.

Figure 4 shows an assay database in accordance with the invention.
Figure 5 shows a baseline data acquisition table for an illustrative multiple analyte assay in accordance with the invention.

Figure 6 shows an assay definition table in accordance with the invention.
Figure 7 shows a discriminant table for an illustrative multiple analyte assay in accordance with the invention.

Figure 8 shows a decision tree view of the illustrative discriminant function table of to Figure 7.

Figure 9 is a flow-chart for a real-time analysis phase of a multiple analyte assay in accordance with the invention.

Figure 10 shows a results table for an illustrative multiple analyte assay in accordance with the invention.

Figure 11 shows a interpretation table for an illustrative multiple analyte assay in accordance with the invention.
Figure 12 is a flow-chart for an interpretation phase of a multiple analyte assay in accordance with the invention Figures 13a through Be show an assay database in accordance with the invention for a specific experimental example.

Figure 14 shows a decision tree view for an illustrative (experimental example) discriminant table.
Figures 15a, 15b, and 15c show individual inhibition assays for IgG, IgA, and IgM
antibodies.

Figures 16a, 16b, and 16c show cross reactivity determinations between IgG, IgA, and IgM assay components.

Figure 17 shows the determination of human IgG concentrations by flow cytometry.
Figure 18 shows the determination of human IgA concentrations by flow cytometry.
Figure 19 shows the determination of human IgM concentrations by flow cytometry.
Figure 20 shows the simultaneous determination of human IgG, IgA, and IgM
concentrations by flow cytometry.

Figure 21 shows the specificity of monoclonal antibody MAB384 binding towards bead immobilized epitope sequences.

Figure 22 shows the specificity of monoclonal antibody MAB384 binding in the presence of soluble epitope containing peptide.

Figure 23 shows the specificity of monoclonal antibody MAB384 binding in the presence of soluble biotin.

Figure 24 shows the detection of anti-Rubella IgG antibodies by a sandwich assay between rubella coated beads and a fluorescent goat anti-human IgG antibody.

Figure 25 shows a calibration assay using serial dilutions of anti-Rubella IgG
antibodies in a sandwich assay between rubella coated beads and a fluorescent goat anti-human IgG
antibody.
Figures 26a and 26b show the simultaneous assay for six anti-ToRCH IgG, and simultaneous assay for the six anti-ToRCH IgM antibodies.

Figure 27 shows the determination of IgG anti-grass allergen activities for six dogs.
Figure 28 shows the determination of IgE anti-grass allergen activities for six dogs.
Figure 29 shows the multiple analyte IgG and IgE screening of dog serum A96324 for io sixteen grass allergens Figure 30 shows the multiple analyte IgG and IgE screening of dog serum A96325 for sixteen grass allergens Figure 31 shows the multiple analyte IgG and IgE screening of dog serum A96319 for sixteen grass allergens Figure 32 shows the multiple analyte IgG and IgE screening of dog serum A96317 for sixteen grass allergens Figure 33 shows the multiple analyte IgG and IgE screening of dog serum A96326 for sixteen grass allergens Figure 34 shows the multiple analyte IgG and IgE screening of dog serum A96323 for sixteen grass allergens Figure 35 shows an antibody pair analysis for use with a human chorionic gonadotropin capture assay.
Figure 36 shows the use of bead linked antibody MAB602 with fluorescently labeled antibody AB633 in a human chorionic gonadotropin capture assay.

Figure 37a and 37b show cross reactivity analyses between components of an anti-hCG
s capture system and an anti-AFP capture system.

Figures 38a and 38b compare the effects of eliminating wash steps in hCG and AFP
capture system assays.

Figures 39a and 39b show the determination of hCG and AFP concentrations in samples and standards using a homogeneous capture assay format.

Figure 40 shows the inhibition of Anti-IgG binding to bead based IgG by soluble IgG
antibodies. Inhibition was determined at five concentrations of soluble IgG, and four IgG
loading levels on the beads.

Figure 41 shows the slope of the inhibition pattern across the four loading levels of IgG
on the beads plotted against the soluble IgG concentration.

Figure 42 shows a five point standard curve derived from inhibition data of the 50 g/mL IgG bead set.

Figures 43a through 43c show DNA detection using a double stranded competitor and a wild-type "B" oligonucleotide probe.
Figures 44a and 44b show DNA detection using a single stranded competitor and a wild-type "B" oligonucleotideprobe.

Figure 45 shows the differentiation by orange and red fluorescence of fourteen bead sets.
Figure 46 shows a titration of a fluorescent oligonucleotide in the presence or absence of an inhibitor. Beads bearing complementary oligonucleotides were used in a capture assay.
Figure 47 shows the inhibition of binding between a fluorescent oligonucleotide and its complementary oligonucleotide bound to a bead. Varying concentrations of complementary and point mutant competitors were used in the determination.

Figure 48 shows the efficacy of inhibitors across fourteen DNA sequence binding sets.
Figure 49 A-D shows the typing of four simulated alleles of DQA1.

Figure 50 A-E shows the typing of five known, homozygous DQA1 alleles.

Figures 51 a through 51 f show the results of an exemplary multiplexed assay according to the invention.

According to the present invention, assay components and methods for the measurement of enzymes, DNA fragments, antibodies, and other biomolecules are provided.
The inventive technology improves the speed and sensitivity of flow cytometric analysis while reducing the cost of performing diagnostic and genetic assays. Further, and of tremendous significance, a multiplexed assay in accordance with the invention enables the simultaneous automated assay of multiple (at least an order of magnitude greater than available in the prior techniques) biomolecules or DNA sequences in real-time.

As those of ordinary skill in the art will recognize, the invention has an enormous number of applications in diagnostic assay techniques. Beadsets may be prepared, for example, so as to detect or screen for any of a number of sample characteristics, pathological conditions, or reactants in fluids. Beadsets may be designed, for example, to detect antigens or antibodies associated with any of a number of infectious agents including (without limitation, bacteria, viruses, fungi, mycoplasma, rickettsia, chlamydia, and protozoa), to assay for autoantibodies 3o associated with autoimmune disease, to assay for agents of sexually transmitted disease, or to assay for analytes associated with pulmonary disorders, gastrointestinal disorders, cardiovascular disorders, and the like. Similarly, the headset may be designed to detect any of a number of substances of abuse, environmental substances, or substances of veterinary importance. An advantage of the invention is that it allows one to assemble a panel of tests that may be run on an individual suspected of having a syndrome to simultaneously detect a causative agent for the syndrome.
Suitable panels may include, for example, a tumor marker panel including antigens such as prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), and other suitable tumor markers; a regional allergy panel including pollen and allergens tested for by allergists of a io particular region and comprising allergens known to occur in that region; a pregnancy panel comprising tests for human chorionic gonadotropin, hepatitis B surface antigen, rubella virus, alpha fetoprotein, 3' estradiol, and other substances of interest in a pregnant individual; a hormone panel comprising tests for T4, TSH, and other hormones of interests;
an autoimmune disease panel comprising tests for rheumatoid factors and antinuclear antibodies and other 1s markers associated with autoimmune disease; a blood borne virus panel and a therapeutic drug panel comprising tests for Cyclosporin, Digoxin, and other therapeutic drugs of interest.

Bead Technology An important feature of the flow cytometric technology and techniques described here is 20 the fabrication and use of particles (e.g., microspheres or beads that make up a beadset). It is through the use of appropriately labeled homogeneous bead subsets, combined to produce a pooled beadset, that the instant multiplexed assay method is practiced. Beads suitable for use as a starting material in accordance with the invention are generally known in the art and may be obtained from manufacturers such as Spherotech (Libertyville, IL) and Molecular Probes 25 (Eugene, OR). Once a homogeneous subset of beads is obtained, the beads are labeled with an appropriate reactant such as a biomolecule, DNA sequence, and/or other reactant. Known methods to incorporate such labels include polymerization, dissolving, and attachment.

A Method for the Multiplexed Assay of Clinical Samples Development of a multiplexed assay for use in accordance with the invention can be divided into three phases: (1) preprocessing, (2) real-time analysis, and (3) interpretation.
During the preprocessing phase, baseline data is collected independently, via flow cytometric techniques, for each of an assay's bead subsets. Baseline data is used to generate a set of functions that can classify any individual bead as belonging to one of the assay's subsets or to a rejection class. During the analysis phase, flow cytometric measurements are used to classify, in real-time, each bead within an exposed beadset according to the aforementioned functions.
Additionally, measurements relating to each subset's analyte are accumulated.
During the interpretation phase the assay's real-time numerical results are associated with textual io explanations and these textual explanations are displayed to a user.

The inventive method allows the detection of a plurality of analytes simultaneously during a single flow cytometric processing step. Benefits of the inventive multiplex assay method include increased speed and reduced cost to analyze a clinical sample.

System Hardware Figure 1 shows, in block diagram form, a system for implementing the inventive multiplexed assay method. Flow cytometer 100 output consists of a series of electrical signals indicative of one or more specified measured characteristics on each bead processed. These measurement signals are transmitted to computer 105 via data bus 110 and interface board 115.
During the preprocessing phase, the signals are used by the computer to generate an assay database. During the real-time analysis phase, the signals are processed by the computer (using the assay database) in accordance with the inventive method to produce a multiplexed/simultaneous assay of a clinical sample.

Flow cytometer 100 operates in a conventional manner. That is, beads are processed by illuminating them, essentially one at a time, with a laser beam. Measurements of the scattered laser light are obtained for each illuminated bead by a plurality of optical detectors. In addition, if a bead contains at least one appropriate fluorescing compound it will fluoresce when illuminated. A plurality of optical detectors within the flow cytometer measure fluorescence at a plurality of wavelengths. Typical measured bead characteristics include, but are not limited to, forward light scatter, side light scatter. red fluorescence, green fluorescence, and orange fluorescence. One of ordinary skill in the use of flow cytometric techniques will recognize that the use of green fluorescent markers or labels can cause cross-channel interference between s optical detectors designed to detect green and orange wavelengths (e.g., approximately 530 nanometers and approximately 585 nanometers respectively). A training set of beads, in combination with standard data manipulation, can correct for this cross-channel interference by providing the physical measurements required for mathematical correction of the fluorescence measurements.

One of ordinary skill will further recognize that many alternative flow cytometer setups are possible. For instance, additional color sensitive detectors could be used to measure the presence of other fluorescence wavelengths. Further, two or more laser beams can be used in combination to illuminate beads as they flow through the cytometer to allow excitation of fluorochromes at different wavelengths.

Computer 105 can be a conventional computer such as a personal computer or engineering workstation. In one embodiment, the computer is a personal computer having an Intel "486" processor, running Microsoft Corporation's "WINDOWS" operating system, and a number of ISA expansion slots.

Interface board 115 is designed to plug into one of the computer's 105 ISA
(Industry Standard Architecture) expansion slots. While the design of an interface board is, in general, different for each specific type of flow cytometer 100, its primary functions include (1) receiving and parsing measurement data signals generated by the flow cytometer's detectors, (2) receiving control parameter status information from the flow cytometer, and (3) sending control parameter commands to the flow cytometer. The precise manner in which these functions are carried out are dependent upon the type (make and model) of the flow cytometer used. In one embodiment, employing a Becton-Dickinson "FACSCAN" flow cytometer (San Jose, CA), the interface board uses control signals generated by the flow cytometer to distinguish measurement -2l data and flow cytometer parameter and control signals. Measured data include forward light scatter, side light scatter, red fluorescence, green fluorescence, and orange fluorescence.
Parameter and control signals include flow cytometer amplifier gain adjustments and status information.

While the design of an interface board 115 for use with the inventive assay method would be a routine task for one skilled in the art of diagnostic medical equipment design having the benefit of this disclosure, an important aspect for any interface board is its ability to accommodate the transmission data rate generated by whatever flow cytometer is used. For io example, the "FACSCAN" flow cytometer can transmit a 16-bit (2 byte) word every 4 microseconds resulting in burst data rates of 500,000 bytes per second.
Microfiche appendix A
provides a detailed source code embodiment of the inventive assay method for use with the "FACSCAN" flow cytometer.

15 Data bus 110 provides a physical communication link between the flow cytometer 100 and the interface board 115. Its physical and electrical characteristics (e.g., data width and bandwidth) are dependent upon the capabilities of the flow cytometer. It is noted that the data bus need not be a totally digital bus. If the flow cytometer does not include analog-to-digital conversion of measured bead characteristics (e.g., light scatter and fluorescence signals), then 20 the data bus must communicate these analog signals to the interface board.
It is then necessary that digital conversion of these signals be provided by either the interface board or another peripheral device before the data is transmitted to the computer 105.

System Software 25 As shown in Figure 2, the software architecture for the inventive assay method can be divided into two parts. A graphical user interface (GUI) 200 provides the means by which a user (1) receives assay results and (2) interacts with the flow cytometer. A
dynamically linked library (DLL) 205 provides the means through which the inventive real-time assay is performed and includes routines necessary to (1) interact with interface board 115 and (2) send and receive 3o information to the flow cytometer 100.

An important aspect of the inventive assay method is that it performs a simultaneous analysis for multiple analytes in real-time. One of ordinary skill in the art of computer software development will realize that real-time processing can impose severe time constraints on the s operational program code, i.e., the DLL 205. For example, the "FACSCAN" flow cytometer can process, or measure, approximately 2,000 beads per second, where each bead is associated with eight 16-bit data values. Thus, to process flow cytometer data in real-time from a "FACSCAN," the DLL should be able to accept, and process, at a consistent data rate of at least 32,000 bytes per second. The need to accommodate this data rate, while also having sufficient io time to perform real-time analysis based on the data, will generally necessitate that some of the DLL code be written in assembly language.

In a current embodiment, the GUI 200 is implemented in the visual basic programming language and the DLL 205 is implemented in C and assembly language programming.
is Microfiche appendix A contains source code listings for one embodiment of the GUI and DLL.
Preprocessing A function of the preprocessing phase is to generate an assay database for use during the real-time analysis of an exposed beadset (clinical sample). Thus, preprocessing is performed 20 prior to combining separately labeled bead subsets to form assay beadsets.
Assay definition, discriminant function definition, and interpretation tables are created at the time an assay beadset is created. Figure 3 shows, in flow chart form, the steps taken during the preprocessing phase.

25 A bead subset is characterized by (1) the analyte it is designed to identify, (2) one or more classification parameters C, ... C,,, and (3) one or more measurement parameters Fml -F.. During the preprocessing phase the classification parameters are used to generate a set of functions, referred to as discriminant functions, that can classify a bead as belonging to one of the assay's subsets or a rejection class. Measurement parameters are used during the real-time analysis phase to determine if a specified analyte is present in the clinical sample being analyzed.

The precise number of individual beads contained in any given subset is relatively unimportant, the only significant criterion being that a sufficient number are used so that a good statistical characterization of the subset's parameters can be achieved during the real-time analysis phase. In a current embodiment, each bead subset contains an equal number of beads.
One of ordinary skill in the field will recognize that the precise number of beads within any given bead subset can vary depending upon many factors including, but not limited to, the io number of analytes an assay beadset is designed to detect, the uniformity of the labeled beads (with respect to each of the measured parameters Cj ... C,,, Fiii1 ... Fes), and the penalty of misclassifying (e.g., making a type 1 or type 2 classification error) a bead during analysis.

During preprocessing, each bead in an unexposed subset is measured by a flow is cytometer 100 and the resulting data values accumulated for later use 300.
For example, if the flow cytometer measures n classification parameters and x measurement parameters, i.e., generates (n + x) values for each bead, data for each of the subset's (n + x) parameters are updated based on each bead's measurements. This data collection step is repeated independently for each subset in the assay's beadset 305. The collection of such data for each of an assay's 20 subsets constitutes an assay's baseline data.

After an assay's baseline data has been collected, a set of discriminant functions are determined 310. During real-time analysis, the discriminant functions are used to classify a bead into one of the assay's bead subsets or a rejection class based solely on the measured 25 classification parameters, CI ... C,,. This step, in principle and practice, is a problem of multi-dimensional classification or cluster analysis. Many prior art techniques and commercial software programs exist to perform this task.

Beads are generally manufactured in large quantities referred to as batches.
Each bead in 30 a batch is of nearly identical size and has substantially the same dye absorption capacity. In light of this manufacturing process, bead subsets can be created using precise dilutions of chosen dyes and, because of their nearly identical size, all classification parameters will exhibit essentially equal variances. By correcting for scaling of the photo-multipliers within a flow cytometer, a linear classification rule can be generated. Further, since there are equal quantities of beads in each subset, the prior probabilities will be equal. This allows use of Fisher's linear discriminant technique to calculate the discriminant functions which define classification boundaries. See, Fisher, "The Use of Multiple Measurements in Taxonomic Problems," Annals of Eugenics, 7, 179-188 (1936). For instance, linear hierarchical discriminant functions may be chosen which are equidistant, in a Euclidean sense, between the centers or centroids of any two io of an assay's bead subsets. Notwithstanding the present example, other types of discriminant functions, such as quadratic functions and those discriminating on more than two classification parameters at once, are also possible.

In addition to the discriminant functions, a set of threshold values are chosen which are is used during the real-time analysis phase to detect the presence of a target analyte. For example, assume measurement parameter F,,,1 is used to detect analyte-A. During preprocessing, the baseline or unexposed value for F,,t1 is measured and accumulated for that subset's beads.
Analyte-A's threshold could then, for example, be set to F, ,I's baseline mean value plus one standard deviation of Fm1's baseline value. One of ordinary skill will recognize that the precise 20 function or value selected for a threshold depends upon the parameter being measured (e.g., its distribution) and the cost of making a classification error (e.g., a type 1 or a type 2 error). It is routine that such values be based on an empirical review of the baseline data.
The important criterion is that the threshold reliably distinguish between the presence and absence of the target analyte in an exposed assay beadset.

After baseline data for each of an assay's bead subsets are collected and discriminant functions and analyte threshold values are established, an assay database is generated 315.

Assay Database As shown in Figure 4, an assay database 400 consists of an assay definition table 405, a discriminant function table 410, a results table 415, and an interpretation table 420. See Figure 4.

The assay definition table 405 defines an assay which, as described above, comprises two or more bead subsets each of which is designed to detect a specified analyte. Each row in the assay definition table describes a bead subset and contains the following entries: (1) assay name, (2) subset name, (3) subset token, (4) baseline values for each of the subset's 1o measurement parameters FmI - F., and (5) test-type token. The subset name entry is a text string identifying the subset by, for example, the type of analyte it is labeled to detect. The subset token is a unique subset identifier. The measurement parameter baseline entries are used during the interpretation phase to associate a numerical result (collected during the real-time analysis of a clinical sample) with a textual output string. Finally, the test-type token identifies which one of a possible plurality of interpretation tests to perform on the collected (real-time) data during the interpretation phase.

The discriminant function table 410 is used to systematically set forth an assay's set of discriminant functions. Each row in the discriminant function table implements a single discriminant function and includes entries for (1) the assay's name, (2) a unique row identifier, (3) one or more classification parameters upon which to evaluate, (4) high and low discriminant values for each of the listed classification parameters, and (5) evaluation tokens which are assigned as a result of evaluating the discriminant function.

The results table 415 is used to store, or accumulate, data on an assay's beadset during the real-time analysis phase of the inventive method and is discussed further in Section 6.2(d).
The interpretation table 420 provides a means to associate text messages with each enumerated assay result and is discussed further in Section 6.2(e).

Preprocessing Example Consider an assay beadset designed to simultaneously detect four analytes:
analyte-A, analyte-B, analyte-C, and analyte-D. Thus, the assay's beadset is comprised of four bead subsets, each labeled for a different analyte. Suppose further that the assay beadset is to be s processed by a Becton-Dickinson Immunocytometry Systems "FACSCAN" flow cytometer.
For each bead processed, the "FACSCAN" measures forward light scatter, side light scatter, red fluorescence, orange fluorescence, and green fluorescence. Let classification parameter CI be forward light scatter, classification parameter C2 be side light scatter, classification parameter C3 be red fluorescence, classification parameter C4 be orange fluorescence, and measurement io parameter F,,,J be green fluorescence. (This notation implies that each bead in a subset is labeled with a green fluorophore bearing, for example, an antibody or dye specifically targeted to that subset's analyte.) After preparing each of the four subsets and before they are combined to form the assay 15 beadset, they are processed by the flow cytometer and their measured data are accumulated:
values for each of the parameters C1, C2, C3, C4, and F.,,1 are recorded for each bead. Each bead subset is similarly processed. Completion of this task constitutes completion of baseline data acquisition.

20 Using baseline data, the assay's beads are clustered in the four-dimensional parameter space defined by C1, C2, C3, and C4. The result of this cluster analysis is that each subset is characterized by a mean ( ) and standard deviation (a) for each of its four classification parameters. See Figure 5. As previously noted, the precise number of individual beads contained in any given bead subset can be calculated by those of ordinary skill in the art. This 25 calculation is required to obtain good statistical characterization of the subset's parameters - e.g., small, or relatively fixed, coefficient of variations for each parameter.

As shown in Figure 6, the assay definition table 405 is comprised of general information relevant to the overall diagnostic function of the assay. For instance, in a genotyping assay, each 30 of the assay's subset's may be assigned a token used for identification:
e.g., token 46 represents WO 97/14028 PCT/1JS96/16198, the bead subset labeled to detect a wildtype coding sequence for a specified gene; subset tokens 21, 50, and 5 represent subsets labeled to detect various mutant type coding sequences for a specified gene(s). Additionally, measurement parameter Fiia1's baseline (in this example the mean) and standard deviation values are listed. Finally, a test-type token is listed. In the current embodiment a test-type token of '0' means an OVER/UNDER interpretation test is to be performed and a test-type token of '1' means a SHIFT interpretation test is to be performed. See Section 6.2(f) for further discussion of these issues.

Discriminate functions are generated by viewing the assay's baseline data graphically in to three dimensions and creating planes to separate the different subset clusters. These "planes"
are created by applying Fischer's Linear Discriminant to the n-dimensional classification parameter space. A populated discriminate function table based on the baseline data of Figure 5 is shown in Figure 7.

is The discriminant function table provides a systematic means of evaluating a series of classification values (Cl, C2, C3, C4) in order to classify a bead. In general bead classification proceeds by entering the discriminant function table at row 0, performing a test on a specified parameter (e.g., C1, C2, C3, or C4) and then, depending upon the result, either classifying the bead or proceeding to another test which involves evaluating a different row in the table. For 20 example, suppose bead A has the following measured classification parameter values: C1 = V1, C2 = V2, C3 = V3, and C4 = V4. Classification of bead A via the discriminant function table of Figure 7 begins as follows (the pseudo-code below would demonstrate to those skilled in the art of programming the logic involved in the classification process):

25 1. Enter table at row 0 with measured values for C1, C2, C3, and C4.
2. If (LOW VALUE = 500) _< (PARAMETER = Cl = VI) <_ (HIGH VALUE = 620) then (result = TRUE), else (result = FALSE).
3. If (result = TRUE) and (TRUE ROW ID # 0), then re-enter table at TRUE ROW
ID, else 4. If (result = TRUE) and (TRUE ROW ID = 0), then (class = TRUE TOKEN).

5. If (result = FALSE) and (FALSE ROW ID # 0), then re-enter table at (row =
FALSE
ROW ID), else 6. If (result = FALSE) and (FALSE ROW ID = 0), then (class = FALSE TOKEN).
7. If (TRUE TOKEN or FALSE TOKEN) = 0, then (class = reject class).
One of ordinary skill will recognize from the above discussion that a discriminant function table embodies a (classification) decision tree. Figure 8 shows this relationship for the discriminant function table of Figure 7 explicitly. A discussion of the discriminant function table as it relates to the real-time processing of an exposed assay beadset is provided in Section io 6.2(d). Once a beadset is preprocessed, the data may be employed in real-time analysis of many assays using that set. One of ordinary skill in the art will also recognize that instead of a decision tree, a bitmap or look up table could be used to classify the bead sets.

Real-Time Analysis Once a collection of bead subsets have been characterized as described above and combined to form an assay beadset, the beadset may be exposed to a test sample. That is, they may be used to analyze a clinical sample. After exposure the beadset is ready for real-time analysis. The real-time analysis phase is initiated by installing the exposed beads into a conventional flow cytometer for processing.

As described above, for each bead processed a flow cytometer 100 generates electrical signals indicative of a plurality of measured parameters, C1 ... C,,, Fml ...
FmX. These values are transmitted to computer 105 via data bus 110 and interface board 115. Values for a bead's classification parameters CI ... Cõ are used to evaluate the assay's discriminant functions, as encoded in a discriminant function table 410, the result of which is an initial classification of the bead into one of the assay's bead subsets or a reject class.

After this initial classification, a bead's measured classification parameter values C, ...
Cõ can be checked against their (Cl ... Cõ) baseline values to determine if it is "reasonable" to classify the bead as belonging to the initially identified class. In a current embodiment, this reasonableness test is implemented by computing the distance between the measured classification parameter values and the mean values obtained during preprocessing. If the measured values for CI ... Cõ for a particular bead are sufficiently distant from the identified subsets baseline values, the bead is assigned to a reject class. Use of this technique allows for s the rejection of beads that were initially misclassified and improves the overall reliability of the analysis.

To ensure proper classification, a preferred embodiment's pooled beadset will include a bead subset which has no bound reactants (e.g., a placebo bead subset) in a known ratio to the 1o beadset's other subsets.
It is noted that when a beadset is comprised of beads manufactured in a single batch, the above described reasonableness test can be incorporated into the linear discriminant functions by creating reject space between all subsets. However, when a beadset is comprised of beads from more than one batch a Euclidean (or similar) distance measure is needed to validate the 15 classification result.

Once a bead is assigned its final classification, the assay's results table 415 is updated to reflect the newly classified bead's measurement parameter values F,,,1 ...
F,,,x. This data acquisition, classification, and update process is repeated for each bead in the assay beadset in 20 real-time. Figure 9 shows, in block diagram form, the general steps performed during the real-time analysis phase of a method in accordance with the invention.

In one embodiment the following data are accumulated in the results table for each class (subset) of bead in the assay: (1) total count of the number of beads detected in the specified 25 class, (2) a running sum for each measurement parameter Fm1 - F., (3) for each measurement parameter the total count of the number of beads in the class whose measurement value is less than the parameter's baseline value, and (4) for each measurement parameter the total count of the number of beads in the class whose measurement value is more than the parameter's baseline value.

Real-Time Analysis Example In the illustrative embodiment introduced in Section 6.2(c), the assay beadset is designed to simultaneously detect four analytes using four classification parameters (Cl represents forward light scatter, C2 represents side light scatter, C3 represents red fluorescence, and C4 represents orange fluorescence) and one measurement parameter (F,,,, representing green fluorescence). After exposing the beadset to a suitable biological sample, it is placed into a flow cytometer 100 which processes each bead (e.g., measures parameters C,, C2, C3, C4, and F,,,1) and transmits to computer 105 signals indicative of these measurements via data bus 110 and interface board 115.

For each bead processed by the flow cytometer, values for C1, C2, C3, and C4 are evaluated in accordance with the discriminant function table shown in Figure 7 to initially classify the bead as belonging to a particular subset, for example, in a genetic analysis intended to detect mutations in the Kras oncogene, the classification could proceed as follows: (1) class 46, Kras CODON 46 WILDTYPE, (2) class 21, Kras CODON 21 MUTANT, (3) class 50, Kras CODON 50 MUTANT, (4) class 5, Kras CODON 5 MUTANT, or (5) a reject class. (See Figure 8 for a decision tree representation of the discriminate function table of Figure 7.) If the bead is initially classified as belonging to any class except the reject class, a reasonableness test is performed on the bead's classification parameter values, C, - C,,. For example, if the bead received an initial classification of class 50 and its C, value is more than two standard deviations away from its mean, the bead is given a final classification of reject. Otherwise the bead's final classification is the same as its initial classification - 50.

If the bead's final classification is other than reject, its F,,,, value is used to update the assay's results table in the following manner (see Figure 10):

1. Identifying, based on the bead's classification token (i.e., subset token 46, 21, 50, or 5), the row in the results table which is to be updated.
2. Incrementing the identified row's COUNT value. The COUNT value reflects the total number of beads of the specified class that have been identified during the analysis.

3. Adding the bead's Fm1 value to the value contained in the row's SUM column.
The SUM
value reflects a running sum of the identified classes measurement values.
4. If the bead's Fiir1 value is greater than Fml's base value (determined during the preprocessing phase, see Figure 6), then incrementing the row's OVER COUNT
value.
The OVER COUNT value reflects the total number of beads of the specified class that have been processed whose Fm1 values are above that of baseline.
5. If the bead's Fm j value is less than Fm1's base value (as determined during the preprocessing phase, see Figure 6), then incrementing the row's UNDER COUNT
value.
The UNDER COUNT value reflects the total number of beads of the specified class that io have been processed whose Fmj values are below that of baseline.
In a preferred embodiment, data (i.e., count, and measured Fmj values) for each bead classified as a reject can also be collected.

Interpretation is Following the real-time classification and accumulation of results as described above, the user may select to see a text based presentation or interpretation of the assay's numerical results.
During the interpretation phase the assay's real-time numerical results are associated with textual explanations. These textual explanations can be displayed to the user.

It is the function of the interpretation table 420 to associate textual descriptions of an assay's possible outcomes with an actual assay's numerical results. Each row in the interpretation table provides the necessary information to make a single interpretation and typically includes entries for (1) the assay's name, (2) a subset token identifying the class or subset on which the interpretation is based, (3) an outcome identifier for the identified subset, (4) a test-type token, (5) high and low discriminant values for each measurement parameter utilized in the identified test, and (6) a text string describing the row's result.

The test-type token identifies which one of a possible plurality of interpretation tests to perform on the collected (real-time) data during the interpretation phase. In a current CA 02227895_1998-01-23 embodiment the test-type token is either '0' or 'P. A value of '0' indicates an OVER/UNDER
interpretation test is to be performed. A value of '1' indicates a SHIFT
interpretation test is to be performed. These tests are defined in the following manner:

OVER/UNDER Test Value = OVER COUNT , and UNDER COUNT' SHIFT Test Value = SUM/COUNT
Baseline FValue where the variables OVER COUNT, UNDER COUNT, SUM, COUNT, and baseline Fm are io described above in Section 6.2(d).

The OVER/UNDER test is generally used for qualitative measurements where the level of reactivity of beads is an indication of the condition or concentration of a biomolecule present in the sample. The shift test is used where the result sought is a determination of the a minimally detectable level of a particular biomolecule. One of ordinary skill will recognize that many other tests could be performed. Examples include ranking, stratification, ratio of means to a standard, or to each other, etc.

In general an interpretation table 420 may associate any number of entries or interpretations (e.g., rows within the table) with a single assay class or bead subset. For instance, bead subset Y could have a single measurement parameter (Fm1) associated with it and this measurement parameter could indicate, depending upon its value, that one or more interpretations are appropriate.

Note, the contents of the interpretation table 420 are generated during the preprocessing phase. This implies that the target assay be understood and that the various assay results be considered prior to construction of multiplexed assays.

Interpretation Example Consider again the assay beadset, introduced above, designed to simultaneously detect four analytes. Figure 11 shows a sample interpretation table for this assay.
Interpretation of the assay's real-time numerical results is initiated by, for example, the user selecting "interpret results" via the inventive method's graphical user interface.

As described above, each bead subset (class) within an assay has an entry or row in the results table, Figure 10. The general procedure for interpreting an assay's real-time numerical results is shown in flow-chart form in Figure 12. In general, each row of the results table is io matched against every row in the interpretation table with the same subset token. If the result of performing the specified test is between the identified row's low and high values, then the associated textual message is displayed to the user. When all rows in the interpretation table for a single results table row have been checked, the next results table row is evaluated. This process is repeated until the every row in the interpretation table has been compared to the is appropriate results table entry.

As a specific example, consider the interpretation of subset 50's (KRAS CODON

MUTANT, see Figure 6) results table entry. The subset's token, 50, is used to identify three rows in the interpretation table (having outcome IDs of 1, 2, and 3) that contain information 20 regarding evaluation of the mutant analyte. For the first identified row, the test-type token indicates a SHIFT type interpretation test is to be performed. Performing this test, as defined above, yields:

1,700,000/
SHIFT Test Value = SUM/ COUNT /1,000 = 10 Baseline F,,, Value 170 Next, the computed SHIFT test value is compared against each interval in the identified rows of the interpretation table. For the row having OUTCOME ID equal to 1, since (LOW
VALUE = 10) - SHIFT Test Value = 10 < (HIGH VALUE = 667) is true, that row's INTERPRETATION entry - "identical complementary strand" - is displayed to the user. This process is repeated for subset 50's remaining two rows in the interpretation table. Further, this process is repeated for each row in the results table.

The result of the interpretation phase is a series of textual messages that describe the s results of the assay. Conclusion of the interpretation phase marks the end of the assay.
Operational Considerations Assay definition, discriminant function definition, and interpretation tables are created at the time an assay beadset is created. Baseline classification data is collected only once for a to given assay. That is, once an assay is defined and its baseline data is obtained, any number of beadsets can be manufactured to perform the analysis. To allow this "sharing"
of baseline data the assay beadset may contain a center or calibration bead subset.

As would be known to those of ordinary skill in the field, a calibration beadset can be is used to adjust any given flow cytometer to a standard. Calibration beadsets are typically processed separately from an assay. Further, calibration is generally performed daily. The purpose of calibration is to adjust the sensitivity of a flow cytometer's photomultipliers to accommodate day to day and machine to machine differences.

20 Unlike prior art calibration techniques which are performed manually, the processing of a calibration beadset and the adjustment of flow cytometer operational parameters (e.g., photomultiplier voltages) is performed under software control automatically.
See microfiche appendix A for embodiment details.

25 Antibody Detection Assays for antibody are widely used in medicine and clinical analysis for an wide variety of purposes, from detection of infections to determination of autoantibody.
The following example illustrates use of the inventive method in an antibody assay and assumes the use of a flow cytometer capable of providing at least five measurements for each bead processed:
30 forward light scatter as classification parameter C1, side light scatter as classification parameter C2, red fluorescence as classification parameter C3, orange fluorescence as classification parameter C4, and green fluorescence as measurement parameter Fmj.

In one method a number of bead subsets, e.g., subsets 1 through 10 (identified as sSl-s sSlO), are prepared, for example, by using a cell sorter to sort a heterogeneous population to collect a homogeneous subset or alternatively, by preparing the beads using tightly controlled specifications to ensure production of a homogeneous subset. Each subset is distinguishable by its characteristic pattern of classification parameters C1, C2, C3, and C4.
The beads in each subset are then labeled with a different antigen such as AgA, AgB, etc. so as to create a io collection of labeled subsets as follows: sSl-AgA, sS2-AgB, sS3-AgC, sS4-AgD, sS5-AgE, sS6-AgF, sS7-AgG, sS8-AgH, sS9-AgI, and sS 10-AgJ.

Antigens AgA through AgJ may be attached to the beads by any of a number of conventional procedures such as by chemical or physical absorption as described by Colvin et is al., "The Covalent Binding of Enzymes and Immunoglobulins to Hydrophilic Microspheres" in Microspheres: Medical and Biological Applications, 1-13, CRC, Boca Raton, FL, 1988;
Cantarero et al., "The Adsorptive Characteristics of Proteins for Polystyrene and Their Significance in Solid-Phase Immunoassays," Anal. Biochem., 105, 375-382 (1980); and Ilium et al., "Attachment of Monoclonal Antibodies to Microspheres," Methods in Enzymol., 112, 67-84 20 (1985).

After attachment of antigen to the beads' surface, aliquots from each subset are mixed to create a pooled or assay beadset, containing known amounts of beads within each subset.
Preferably, the pooled set is prepared with equal volumes of beads from each subset, so that the 25 set contains about the same number of beads from each subset.

The assay beadset may then be incubated with a fluid sample of interest, such as serum or plasma, to test for the presence of antibodies in the fluid that are reactive with antigens on the beads. Such incubation will generally be performed under conditions of temperature, pH, ionic 30 concentrations, and the like that facilitate specific reaction of antibodies in the fluid sample with antigen on the bead surface. After a period for binding of antibody, the beads in the mixture are centrifuged, washed and incubated (again under controlled conditions) for another period of time with a "secondary" antibody such as, for example, fluorescein labeled goat anti human immunoglobulin. The secondary antibody will bind to and fluorescently label antibodies bound to antigen on the beads. Again after washing (or without washing), the beads are processed by the flow cytometer and the four classification parameters forward light scatter , side light scatter, red fluorescence, and orange fluorescence are measured and used to identify the subset to which each bead in the assay headset belongs. A simultaneous measurement of green fluorescence (measurement parameter) for each bead allows one to determine whether the bead has antibody io bound to it. Because the subset to which a bead belongs is correlated with the presence of a particular antigen, e.g., sS 1 -AgA, one may readily determine the specificity of the antibody bound to a bead as a function of the subset to which it belongs.

Experimental Example is Three different antigen-antibody pairs were used in a multiplex experiment demonstrating the ability to detect the presence or absence of several antibodies in a single sample. Antigens were coupled to latex microspheres via carbodiimide coupling, and the corresponding antibodies were fluorescently labeled with fluorescein isothiocyanate (green fluorescence - F,,,). Each antigen was coupled to a unique microsphere.
Baseline data for the 20 fluorescent antibodies and antigen-microsphere complexes used in this experiment are shown in Figure 13a. Baseline data for the three bead subsets of Figure 13a are given in Figure 13b.
The absence of fluorescence (C2 and C3) immediately discriminates the clear beads (subset 50) from beads in the other two subsets. Subsets 45 and 50 were further discriminated 2s by side light scatter (CI) and red fluorescence (C3). Linear discriminant functions based on these observations and created as described in Section 6.2(c); are shown in Figure 13c.
Accepting only clear beads with side light scatter (CI) within 0.25 standard deviations of the mean, doublets (two beads stuck together) were eliminated from the analyses.
The remaining beads were classified by red fluorescence (C3) at a midpoint of 59.6. A
decision tree based on 30 the discriminant function table (Figure 13c) is shown in Figure 14.

In this experiment, each of four samples (e.g., blood serum from four patients) contained all three antigen-microsphere complexes and either 1 or 2 different fluorescent antibodies in PBS buffer. After addition of the antibodies, the reactions were incubated at room temperature for 45 minutes, and then analyzed on the "FACSCAN" using side light scatter (C1), orange fluorescence (C2), and red fluorescence (C3) as classification parameters.
Green fluorescence was used as the measurement parameter (F,,,); an increase in green fluorescence by 30-fold indicates a specific interaction between an antigen and its corresponding fluorescinated antibody. In other words, if a subset's mean measured F,,, value is greater than 30-fold times that io subset's baseline F,,, value, then the target analyte is determined to be present. These "interpretive" observations are embodied in the interpretation table shown in Figure 13d.

Once the assay database was built, it was tested by running 5,000 beads from each bead subset individually through the system. After rejecting 23.8% of the beads as doublets, the is remaining crimson beads (subset 18) were classified with 99.88% accuracy.
Dark red beads (subset 45) were classified with 99.96% accuracy with 22.9% rejected as doublets. Clear beads (subset 50) were classified with 100% accuracy with 9.4% of the beads rejected as doublets.

The three bead subsets were pooled to form an assay beadset and divided into 4 sample 20 tubes and processed by the system shown in Figure 1. The contents of each sample and the mean measured fluorescence (Fm) for each bead subset are listed in Figure 13e.
The inventive method correctly identified the antibody or antibodies present in each sample.

An Experimental Refinement 25 In an alternative embodiment using a C4 (e.g., orange fluorescence) labeled reactant as a classification parameter, a variety (for example five) of protein antigens are employed. Bead subsets are first generated based on differences in one or more of C1, C2, and C3. Next, a selected antigen labeled with Cy3NHS (an orange fluorophore) is bound to the beads in each subset. To minimize the measured orange fluorescence coefficient of variation for each bead 30 subset, the beads are sorted with a high speed cell sorter so that only a narrow range of antigen (orange fluorophore) is found on each bead within a subset. Care should be taken to select or prepare the beadset so that different C4 values are measured/obtained for each of the (e.g., five) different antigens used. In other words, the measured intensity of C4 for AgA
should differ from the measured intensity of C4 from AgB, etc. To ensure that uniformity is achieved, saturation binding with fluoresceinated monoclonal antibody is tested - each bead ought to have restricted ranges of both orange and green fluorescence. While the construction of beadsets by this method is more laborious, the increase in measurement precision may be useful and will allow the sampling of fewer beads to arrive at a suitable determination of antibody concentration.

io The assays previously mentioned measure any antibody with specificity for antigen upon an appropriately labeled bead. The antigen can be quite simple or rather complex and thus, the inventive methods can measure a highly restricted antibody or a broad array of antibodies. For example, a hexapeptide just large enough to bind to a monoclonal antibody can be employed as antigen or a large protein with many epitopes can be used. One of ordinary skill will recognize that the level of antibody eventually found associated with the bead (Fiii1) is a function of the number of epitopes per bead, the concentration of epitopes, the amount of antibody and the affinity of the antibody and the valence of the antibody-antigen interaction.

Displacement Assays Assays for many substances in a clinical laboratory are based on the interference with specific ligand-ligate or antigen-antibody interactions. In these assays, one member of the ligand-ligate pair is labeled with the Fm fluorophore and one member is immobilized on the beads. Soluble, unlabeled material (analyte) which may be ligand or ligate, is added to the reaction mixture to competitively inhibit interaction of the labeled component with the immobilized component. It is usually not important which member of the pair is labeled and which is immobilized; however, in certain assays, functional advantages may dictate the orientation of the assay.

In an exemplary assay of this type, each bead subset is modified with an antigen. The 3o antigen-coated beads are then reacted with an Fm labeled antibody specific for the antigen on the bead surface. Subsequent addition of a test fluid containing soluble analyte (inhibitor) will displace the F,,, labeled antibody from the beads in direct proportion to the concentration of the soluble analyte. A standard curve of known analyte concentrations is used to provide accurate quantification of analyte in the test sample.

One of ordinary skill will recognize that the time necessary to achieve equilibrium may be quite lengthy due to the kinetics and association constant of the interaction. To lessen the time required for the assay, the fluid containing the beadset may be subjected to dissociating conditions such as a change in pH, ionic strength or temperature, after mixture of the beadset io with the sample to be tested. Alternatively, the F,,, labeled component may be added to the beadset after addition of the test sample. In either case, it is not necessary for equilibrium to be achieved to determine analyte concentration if the kinetics and linearity of the assays have been established.

Additional Experimental Examples The following series of experimental examples illustrates how the above referenced techniques can be used in practice in effective diagnostic assays. In one embodiment for example, a competitive inhibition analysis is used to quantitate levels of selected analytes, here IgG, IgA, and IgM. A second experimental refinement demonstrates the utility of multiplexed assays in epitope mapping of a monoclonal antibody. In one embodiment, that approach involved the use of antibody detection technology using a fluoresceinated monoclonal antibody in combinatorial epitope screening (e.g. of peptide libraries) to map a particular epitope to which a monoclonal antibody of interest bound, together with a displacement (competitive inhibition) aspect to demonstrate the specificity of the assay. Also described is a ToRCH
assay for screening of human serum for antibodies to a number of infectious agents known to pose special hazards to pregnant women. Allergy screening is exemplified by detection of serum IgE against a panel of grass antigens. Yet an additional experimental example reflects the ability of the multiplexed assay in pregnancy testing, e.g. in testing for hormones or other analytes commonly elevated during pregnancy. Each of these examples is set forth below.

Simultaneous competitive inhibition assay of human immunoglobuling G, A and M
levels in serum This example illustrates the determination of multiple analyte levels in a liquid sample simultaneously using competitive inhibition analysis. The use of a competitive inhibition assay to accurately determine analyte levels in liquid solutions is a commonly used format for many analyte assays. The uniqueness of this assay is the simultaneous determination of three distinct serum proteins at the same time in the same tube from one serum sample.

Immunoglobulins G, A and M are three distinct serum proteins whose levels are io determined by a number of genetic and environmental factors in human serum.
As changes to these levels may indicate the presence of disease, clinicians often request assay determinations of IgG, A and M using conventional techniques. The most common technique is nephelometry that depends upon the absorption of light by precipitates formed between these immunoglobulins and antibodies made in animals to the human immunoglobulins.
As these is immunoglobulins are present in human serum at fairly high levels, this type of assay is sufficient. Nephelometry however suffers from a number of limitations including the need for large quantities of reagents, long reaction times for precipitation to equilibrate and an inability to perform more than one reaction per tube or sample.

20 Three competitive inhibition assays are described, one for human IgG, one for human IgM and one for human IgA using three Differentially Fluorescent Microspheres (DFM). Each assay consists of a DFM coated with the immunoglobulin of choice and a polyclonal, goat anti-human Ig labeled with a green fluorescent molecule (Bodipy). In the absence of inhibitor, the Bodipy -antibody causes the immunoglobulin (Ig) coated microsphere to emit green 25 fluorescence (Fm). In the presence of inhibitor (soluble Ig), the green signal is reduced. Each assay is balanced to reflect a sensitivity range near the physiological level of the Ig in question at a 1:500 dilution of human serum. Once balanced, the three assays were combined into a multiple analyte format and assayed simultaneously using flow cytometry.

Antibody labeling: Goat anti-human IgG, goat anti-human IgA, and goat anti-human IgM
antibodies (Cappel Division, Organon Teknika. Durham, NC) were labeled with Bodipy FL-CASE (Molecular Probes, Inc., Eugene. OR) using methods described by the manufacturer of the Bodipy succinymidyl ester. The resulting Bodipy labeled antibodies were stored in PBS
s containing 1 mg/mL BSA as stabilizer.

Antigen conjugation to microspheres: Four DFM (5.5 M carboxylate, Bangs Laboratories, Inc.
(Carmel, IN), dyed by Emerald Diagnostics, Inc. (Eugene, OR)) were conjugated separately to human IgG, human IgA, human IgM (Cappel Division, Organon Teknika, Durham, NC) and io BSA with a two-step EDC coupling method (Pierce Chemicals, Rockford, IL) using sulfo-NHS
to stabilize the amino-reactive intermediate. 100 L of each bead type (4.2 x 107 microspheres) was activated for 20 minutes in a total volume of 500 tL containing 500 4g of EDC and Sulfo-NHS in 50 mM sodium phosphate buffer, pH 7Ø The microspheres were washed twice with 500 pL PBS, pH 7.4 using centrifugation at 13,400 x g for 30 seconds to harvest the is microspheres. Activated, washed beads were suspended in 250 tL of a 0.05 mg/mL solution of protein in PBS, pH 7.4. After 1 hour, the microspheres were blocked by addition of 250 .iL of TM
1.0 mg/mL BSA, 0.02% Tween. 0.2 M glycine, in PBS, pH 7.4 and incubated for an additional 30 minutes. Protein coated microspheres were washed twice with 500 L 0.02%
Tween 20, 1 mg/mL BSA in PBS, pH 7.4 (PBSTB). and stored in PBSTB at approximately 3,000,000 20 microspheres/mL. Microsphere concentrations were determined using a hemacytometer.

Determination of appropriate ranges of quantitation for each Ig assay: The normal range of human Ig levels in serum as reported in Clinical Chemistry: Principles and Technics, 2nd Edition, Edited by R.J. Henry, D.C. Cannon and J.W. Winkleman are 569-2210 mg/dL for IgG, 25 51-425 mg/dL for IgA and 18-279 mg/dL for IgM. Each inhibition assay was designed to be sensitive to inhibition across these ranges.

Single analyte assay: 10 tL of dilutions of a serum calibrator with known Ig levels (Kamiya Biomedical, Thousand Oaks, CA) was first mixed with 10 L of Ig loaded microspheres 30 containing 7,500 beads. Next, 10 L of the Bodipy-labeled Goat Anti-Ig was added and the mixture incubated at ambient temperature for 30 minutes. The mixture was diluted to 300 gL in PBSTB and assayed by flow cytometry. For IgG, the Bodipy-labeled goat anti-hIgG was used at 30 gg/mL. For IgA, the Bodipy-labeled goat anti-hIgA was used at 8 gg/mL.
For IgM, the Bodipy-labeled goat anti-hlgM was used at 2.5 gg/mL.
Cross reactivity assay: Equivalent amounts of each of the four protein loaded microspheres were mixed to produce a bead mixture. 10 gL of the bead mixture (7,500 microspheres) was mixed with 10 gL of diluted serum calibrators of known Ig level. The assay was initiated by addition of 10 gL of one of the Bodipy-labeled antibodies "spiked" with a small quantity of soluble Ig io antigen to alleviate the "hook effect". The mixtures were incubated for 30 minutes, diluted to 300 gL in PBSTB and assayed by flow cytometry. As before for the single analyte assay, the Bodipy-labeled goat anti-hIgG was used at 30 gg/mL. For IgA, the Bodipy-labeled goat anti-h1gA was used at 8 gg/mL. For IgM, the Bodipy-labeled goat anti-hlgM was used at 2.5 gg/mL. The quantities of antigen "spikes" were 1.6 gg/mL for IgG, 0.6 pg/mL
for IgA and 0.4 is gg/mL for IgM.

Multiple analyte assay: Equivalent amounts of each of the four protein loaded microspheres were mixed to produce a bead mixture. 10 gL of the bead mixture (7,500 microspheres) was mixed with 10 gL of diluted serum calibrators of known Ig level as well as three other calibrator 20 sera of known Ig level to serve for this purpose as unknowns. The assay was initiated by addition of 10 gL of a mixture of the three Bodipy-labeled antibodies "spiked"
with a small quantity of the three soluble Ig antigen to alleviate the "hook effect". The mixtures were incubated for 30 minutes, diluted to 300 gL in PBSTB and assayed by flow cytometry. As before, the Bodipy-labeled goat anti-hIgG was used at 30 gg/mL. For IgA, the Bodipy-labeled 25 goat anti-hIgA was used at 8 gg/mL. For IgM, the Bodipy-labeled goat anti-hIgM was used at 2.5 gg/mL. The quantities of antigen "spikes" were 1.6 gg/mL for IgG, 0.6 gg/mL for IgA and 0.4 gg/mL for IgM.

Results IgG single analyte assay: Results of the single analyte inhibition analysis for IgG level is shown in Table 1 and Figure 15A. This assay was designed to be most sensitive to inhibition in the anticipated range of IgG in human serum at a 1:500 dilution. In Figure 15A, the area of the s inhibition curve between the dotted lines, left and right, cover the range of sensitivity. In this case, the inhibitor was known amounts of human IgG from a serum calibrator diluted into human serum containing no IgG, IgA or IgM. Dilutions of the calibrator were then diluted 1:500 in PBSTB and included as inhibitor in the assay. The Bodipy-labeled anti-hIgG was used at 30 g/ml, in PBSTB. 7,500 microspheres were used in this experiment and 250 were counted io by flow cytometry. Note that as the amount of soluble IgG increased, the degree of inhibition as monitored by the MIF of F. increased proportionally until saturation of the system was achieved. On the other end of the inhibition curve note that the lower levels of soluble inhibitor caused an elevation in the MIF of F,,, as compared with the negative control (human serum with no Ig). This "hook effect" is common in immunoassay and can be adjusted up or down the 15 inhibition curve by adjusting both the amount of antibody and antigen in the soluble portion of the assay. The "hook effect" was most prominent in the IgG assay due to the higher concentrations of both antigen and antibody per microsphere. This was necessary as IgG is found in serum at higher concentrations than IgA or IgM.

20 IgA single analyte assay: Results of single analyte inhibition analysis for IgA level is shown in Table 1 and Figure 15B. This assay was designed to be most sensitive to inhibition in the anticipated range of IgA in human serum at a 1:500 dilution. In Figure 15B, the area of the inhibition curve between the dotted lines, left and right, cover the range of sensitivity. In this case, the inhibitor was known amounts of human IgA from a serum calibrator diluted into 2s human serum containing no IgG, IgA or IgM. Dilutions of the calibrator were then diluted 1:500 in PBSTB and included as inhibitor in the assay. The Bodipy-labeled anti-hIgA was used at 8 g/mL in PBSTB. 7,500 microspheres were used in this experiment and 250 were counted by flow cytometry. Note that as the amount of soluble IgA increased, the degree of inhibition as monitored by the MIF of F,,, increased proportionally until saturation of the system was 3o achieved. On the other end of the inhibition curve note that the lower levels of soluble inhibitor cause a slight elevation in the MIF of Fm as compared with the negative control (human serum with no Ig). The "hook effect" was much less pronounced for both IgA and IgM
due to their lower concentrations in serum.

s IgM single analyte assay: Results of single analyte inhibition analysis for IgM level is shown in Table 1 and Figure 15C. This assay was designed to be most sensitive to inhibition in the anticipated range of IgM in human serum at a 1:500 dilution. In Figure 15C, the area of the inhibition curve between the dotted lines, left and right, cover the range of sensitivity. In this case, the inhibitor was known amounts of human IgM from a serum calibrator diluted into io human serum containing no IgG, IgA or IgM. Dilutions of the calibrator were then diluted 1:500 in PBSTB to be included as inhibitor in the assay.. The Bodipy-labeled anti-hIgM was used at 2.5 pg/mL in PBSTB. 7,500 microspheres were used in this experiment and 250 were counted by flow cytometry. Note that as the amount of soluble IgM increased, the degree of inhibition as monitored by the MIF of F. increased proportionally until saturation of the system 15 was achieved. On the other end of the inhibition curve note that the lower levels of soluble inhibitor cause a slight elevation in the MIF of Fm as compared with the negative control (PBS
with no added IgM). The "hook effect" is much less pronounced for both IgA and IgM due to their lower concentrations in serum.

20 Cross reactivity analysis: To determine the cross-reactivity of the various assay components, a multiple analyte assay was performed using only one of the three Bodipy-labeled antibodies.
Equivalent numbers of the IgG, IgA, IgM and BSA beads were mixed to make a GAM
mixed bead set. To 10 .tL of the bead set (7,500 microspheres) was added 10 L of dilutions of the calibrator containing IgG, IgA and IgM. The multiple analyte assay was then performed using 25 only one of the Bodipy-labeled anti-IgG, IgA or IgM preparations rather than a mixture. Table 2 and Figures 16A, 16B, and 16C show the results of these assays. Results indicated that Anti-IgG-Bodipy only reacted with DFM-IgG Bodipy and not the IgA or IgM beads. No cross-reactivity with IgA or IgM was noted and the assay was validated for further multiple analyte analysis. Also added to this analysis was the antigen "spike". By adding a small amount of 30 soluble antigen to the probe antibody solution the "hook effect' can be minimized. Note in the IgG cross-reactivity experiment that the MIF of F,,, for negative control is higher than the lowest concentration of inhibitor. By spiking the experiment with 1.6 g/mL IgG the hook effect has no effect at the lower end of inhibitor range leading to a more accurate assay over the entire dynamic range.
GAM simultaneous analysis: Equivalent numbers of the IgG, IgA, IgM and BSA
beads were mixed to make a GAM mixed bead set. To 10 .tL of the bead set (7,500 microspheres) was added 10 L of dilutions of the calibrator containing IgG, IgA and IgM. Also included were several additional calibrators that served as unknowns for the demonstrative purpose of this to assay. The multiple analyte assay was then initiated by adding 10 L of a mixture of the Bodipy-labeled anti-IgG, IgA and IgM plus the soluble Ig "spikes". After a 30 minute, room temperature incubation the reaction mixture was diluted to 300 L and 1000 microspheres counted by flow cytometry. Tables 3-5 and Figures 17-19 show the results of these assays. For each of the inhibition curves produced, a polynomial trendline was used as a non-linear is regression analysis. The fit of this trendline to the data was demonstrated by the R2 correlation factor (1.0 is a perfect fit). The factors of the polynomial formula were used to predict the quantity of inhibitor in each dilution of calibrator and "unknown" serum. The differences between the predicted inhibitor quantities and actual amounts were also included in Tables 3-5.
Results indicate that this multiple analyte inhibition assay can determine the level of these 3 20 serum proteins with an error of less than 10 %. Coefficients of variation (CV) between the triplicate data points indicated that the assay was highly precise (no CV
greater than 6%).
Limits of quantization for each assay were 400 - 3000 mg/dL for IgG, 60- 455 mg/dL for IgA, and 36 - 272 mg/dL for IgM. Figure 20 shows the results of the three assays graphically represented on the same graph as all three assays were performed at the same time in the same 25 tube.

A multiple analyte, competitive inhibition assay for human serum IgG, IgA, and IgM
levels has been developed. This assay, that allows the simultaneous assay of these three protein levels in serum diluted 1:500, demonstrated excellent sensitivity, precision and accuracy.

TABLE 1: Single analyte inhibition assays IgG MIF of IgA MIF of IgM MIF of Tube# mg/dL Fm mg/dL Fm mg/dL Fm 2 0.026 1500 0.0040 1645 0.0024 1794 3 0.11 1460 0.016 1729 0.010 1929 4 0.42 1512 0.064 1734 0.038 1921 1.7 1426 0.26 1733 0.15 1815 6 6.8 1619 1.02 1747 0.61 1829 7 27.1 1684 4.1 1746 2.4 1833 8 108 1943 16.4 1788 9.8 1807 9 163 1898 24.6 1813 14.7 1792 244 1885 36.9 1806 22.0 1723 11 366 1624 55.3 1703 33.0 1704 12 549 1456 83.0 1391 49.6 1446 13 824 998 125 971 74.3 1267 TABLE 2: Cross-reactivity analysis in multiple analyte assay 1) GAM Beads reacted with anti-IgG -Bodipy @ 30 g/mL + Ag spikes.

hu IgG Bead 1- hu IgA Bead 2- hu IgM Bead 3- Bead 4-MIF MIF MIF MIF
Tube mg/dL HuIgG mg/dL HuIgA mg/dL HuIgM BSA

2 400 1702 60.5 4 36.1 7 5 3 561 1463 84.7 4 50.6 5 5 4 785 1218 119 3 70.8 4 5 1099 880 166 3 99.2 3 5 2) GAM Beads reacted with anti-IgA -Bodipy @ 8 gg/mL + Ag spikes.

12 400 2 60.5 1455 36.1 2 3 13 561 8 84.7 1225 50.6 1 3 14 785 3 119 930 70.8 1 3 1099 2 166 605 99.2 1 3 3) GAM Beads reacted with anti-IgM -Bodipy @ 2.5 gg/mL + Ag spikes.
22 400 3 60.5 9 36.1 1284 2 23 561 3 84.7 2 50.6 1135 2 24 785 3 119 2 70.8 1011 2 25 1099 2 166 2 99.2 776 2 TABLE 3: Multiple analyte IgG inhibition data hIgG MIF Average MIF Calculated %
Tube # mg/dL of Fm MIF CV mg/dL Difference 2 0 1943 1926 0.6% na na 400.4 1737 1772 1.7% 399.3 0.3%

8 560.6 1529 1471 3.4% 566.9 -1.1%

11 784.8 1163 1236 4.4% 775.3 1.2%

14 1099 867 862 0.8% 1102.5 -0.3%

17 1538 726 691 3.9% 1556.1 -1.2%

2154 575 580 1.1% 2126.3 1.3%

23 3015 466 468 1.5% 3025.1 -0.3%

"UNKNOWNS"

26 446 1657 1657 1.7% 411.3 7.8%
29 1243 737 763 3.8% 1316.8 -5.9%
32 3045 486 476 1.9% 2947.1 3.2%
TABLE 4: Multiple analyte IgA inhibition data hIgA MIF Average MIF Calculated %
Tube # mg/dL of Fm MIF CV mg/dL Difference 2 0 1941 1952 0.4% na na 60.5 1664 1665 0.2% 60.5 0.0%

8 84.7 1391 1307 5.3% 84.7 0.0%

11 118.6 974 1051 5.9% 118.6 0.0%

14 166.1 595 606 1.4% 166.1 0.0%

17 232.5 426 400 5.1% 232.6 0.0%

325.5 280 287 2.9% 325.4 0.0%

23 455.7 198 195 1.2% 455.7 0.0%

"UNKNOWNS"

26 65 1483 1504 3.1% 68.2 -4.9%

29 187 457 477 6.1% 197.2 -5.5%

32 454 199 201 5.9% 445.4 1.9%

TABLE 5: Multiple analyte IgM inhibition data hIgM MIF Average MIF Calculated %
Tube # mg/dL of Fm MIF CV mg/dL Difference 2 0 1615 1605 1.8% na na 36.1 1312 1328 1.0% 35.7 1.2%

8 50.6 1182 1155 1.8% 52.9 -4.6%

11 70.8 994 1035 3.2% 68.1 3.9%

14 99.2 733 735 0.9% 100.7 -1.5%

17 138.8 585 546 5.4% 138.3 0.4%

194.4 414 419 1.0% 194.0 0.2%

23 272.1 339 315 5.6% 272.4 -0.1%

"UNKNOWNS"

26 40 1248 1241 1.9% 42.8 -7.0%

29 113 621 635 4.7% 116.2 -2.8%

32 268 315 306 3.9% 281.0 -4.9%

Epitope Mapping of a Monoclonal Antibody using Flow Cytometry.
This example demonstrates the screening of combinatorial chemistry products for a biologically active molecule. The generation of random chemical products for empirical discovery of biologically significant molecules is a method that holds great promise for progress in numerous disciplines of science including biology, pharmacology and medicine. One general problem with the technique is the screening of large numbers of unique molecules for a specific activity. Screening methods are required that provide high throughput levels of screening with io adequate specificity and sensitivity for detection of the biological event in question.

An experiment was designed to demonstrate the screening of peptides for the epitope of a monoclonal antibody. A monoclonal antibody (MAB 384) was chosen that was produced using the spleen cells of a mouse hyper-immunized with a defined peptide (amino acid 67-74) is from the amino acid sequence of human myelin basic protein (MBP). Using the amino acid sequence of this region of MBP, nine overlapping octapeptides were synthesized that covered the predicted epitope. To the carboxyl terminal end of each peptide, glycine-lysine-biotin residues were added. Nine Differentially Fluorescent Microspheres (DFM) were each coated with avidin and one unique peptide of the set was linked through the avidin-biotin interaction to 20 one unique member of the bead set. This resulted in a set of microspheres that contained nine members each carrying a unique peptide either flanking or representing the monoclonal antibody's epitope. The bead carrying the epitope peptide was detected using the MAB 384 antibody labeled with a green fluorescent tag in a multiple analyte analysis.
The detection was shown to be specific for the peptide in question by competitive inhibition and was not affected by high levels of free biotin.

Antibody labeling: MAB 384 (Chemicon International, Inc., Temecula, CA) was labeled with Bodipy FL-X (Molecular Probes, Inc., Eugene, OR) using methods described by the manufacturer of the Bodipy succinymidyl ester. Absorbance at 280 nm and 504 nm revealed io that the resulting Bodipy-labeled antibody had a Bodipy to protein ratio of 3.31 and was stored in PBS containing 1 mg/mL BSA as stabilizer.

Avidin conjugation to microspheres: Nine distinctly dyed DFM (5.5 M, Bangs Laboratories.
Inc. (Carmel, IN), dyed by Emerald Diagnostics, Inc. (Eugene, OR)) were conjugated separately TM
to Neutravidin (deglycosylated avidin) with a two-step EDC coupling method (Pierce Chemicals, Rockford, IL) using sulfo-NHS to stabilize the amino-reactive intermediate. 20 tL
(8.4 million microspheres) of each bead type was activated for 20 minutes in a total volume of 100 L containing 500 .&g of EDC and Sulfo-NHS in 50 mM sodium phosphate buffer, pH 7Ø
The microspheres were washed twice with 100 tL PBS, pH 7.4 using centrifugation at 13,400 x g for 30 seconds to harvest the microspheres. Activated, washed beads were suspended in 50 L
of a 0.25 mg/mL solution of Neutravidin in PBS, pH 7.4. After 2 hours, the microspheres were blocked by addition of 50 L of 0.2 M glycine, 0.02% Tween 20 in PBS, pH 7.4 and incubated for an additional 30 minutes. Protein coated microspheres were washed twice with 100 L
0.02% Tween 20, 1 mg/mL BSA in PBS, pH 7.4 (PBSTB) and stored in PBSTB at approximately 3,000,000 microspheres/mL as determined by hemocytometer count.

Peptide attachment to microspheres: Each of the nine DFM conjugated to Neutravidin were treated separately with one of the nine biotinylated peptides. 10 L of biotinylated peptides at 100 - 200 ng/mL was mixed with 10 p.L of microspheres and reacted for 5 minutes followed by 2 x 100 g L washes in PBSTB. The peptide loaded microspheres were suspended in 20 L of PBSTB.

Single analyte assay: 10 L of each of the peptide loaded microspheres was reacted with 10 gL
of the Bodipy-labeled MAB 384 at 15.5 jig/mL in PBSTB for 1 hour, diluted to 300 L in PBSTB and assayed using flow cytometry. Negative controls included the microspheres without peptide and with the Bodipy MAB 384.

Multiple analyte assay: 10 gL of each of the 9 peptide loaded microspheres was mixed to io produce a bead set. 10 L of the set was reacted with 10 L of the Bodipy-labeled MAB 384 at 15.5 g/mL in PBSTB for 1 hour, diluted to 300 L in PBSTB and assayed using flow cytometry. Negative controls included the microsphere set without peptide and treated with the Bodipy MAB 384.

is Competitive inhibition with soluble peptide: 10 L of each of the 9 peptide loaded microspheres was mixed to produce a bead set. 10 L of the Bodipy-labeled MAB 384 at 15.5 g/mL in PBSTB was reacted with 10 L of soluble peptide containing the epitope sequence HYGSLPQK
(SEQ ID NO. 1) at 10 g/mL and incubated for 1 hr. The microsphere set was then treated with peptide absorbed Bodipy-labeled MAB 384 at 15.5 g/mL for 1 hour, diluted to 300 L in 20 PBSTB and assayed using flow cytometry.

Examination of the effects of free biotin: 10 gL of each of the 9 peptide loaded microspheres was mixed to produce a bead set. 10 L of the mixture was reacted with 10 L
of 10 g/mL
free biotin and incubated for 1 hr. The microsphere set was then treated with Bodipy-labeled 25 MAB 384 at 15.5 g/mL for 1 hour, diluted to 300 L in PBSTB and assayed using flow cytometry.

Results Description of peptides to be screened: The amino acid sequence upstream and downstream 30 from the epitope of monoclonal antibody MAB 384 (amino acid 67-74, YGSLPQ, SEQ ID NO.

2) was determined using the published amino acid sequence (Roth, H.J., et al., J. Neurosci. Res..
17, 321-328, 1990). The table below shows the amino acid sequence of the nine overlapping peptides produced for the screening assay. Note that to the carboxy-terminal end of all peptides was added a glycine (G)-lysine (K)-biotin.
1 GLCNMYKDGK-biotin 2 MYKDSHHPGK-biotin 3 SHHPARTAGK-biotin 4 ARTAHYGSGK-biotin 5 HYGSLPQKGK-biotin 6 LPQKSHGRGK-biotin 7 SHGRTQDEGK-biotin 8 TQDENPVVGK-biotin 9 NPVVHFFKGK-biotin Single vs. multiple analyte analysis: Each of the nine DFM coated with Neutravidin was reacted for 5 minutes with one of the nine biotinylated peptides diluted to 250 ng/mL
in PBS. For single analyte analysis, each separate microsphere was reacted with Bodipy-labeled MAB 384 at io 15.5 gg/mL for 60 minutes and the mixture assayed using flow cytometry. The Mean Intensity of Fluorescence (MIF) of the green fluorescence channel (F,,,) is shown for each peptide-bead as the darker set of bars in Figure 21. The darkest bars represents single analyte analysis of each bead in the absence of peptide as a negative control.

is For multiple analyte analysis, the nine bead-peptides were mixed and then reacted with Bodipy-MAB 384 at 15.5 g/mL. After 60 minutes, the mixture was assayed using flow cytometry and results (MIF of Fm) are also shown in Figure 21. Both assays minus added peptide are shown as a negative control. Results indicated that peptide #5 contained the epitope for MAB 384. Peptides #4 and #6 although containing 3 of the epitope's amino acids showed 20 little reactivity. The multiple and single analyte assays provided identical results. Numerical data is shown in Table 6.

Competitive inhibition using soluble epitope peptide: To further demonstrate the specificity of the assay, soluble peptide containing the epitope (#5) was used to inhibit the reaction shown in Figure 21. A 10 L aliquot of the Bodipy-labeled MAB 384 was mixed with an equal volume of the epitope containing peptide (HYGSLPQK) at 10 g/mL. After 1 hour the mixture was reacted with 10 L of the bead mixture for 1 hour and assayed by flow cytometry. Results shown in Figure 22 reveal that the reaction was significantly inhibited to a MIF of Fm of 53.
Numerical data for the inhibition assay is shown in Table 7.

Effects of free biotin: The high avidity of the biotin-avidin interaction makes it unlikely that the io various peptides could be released or exchanged from microsphere to microsphere, To demonstrate that such a release or exchange does not occur under strenuous conditions the following experiment was performed. A 10 L aliquot of free biotin at 10 g/ml, (40 gM) was incubated with 10 L of the bead-peptide mixture for 1 hour and then the microspheres reacted with the MAB 384 Bodipy at 15.5 g/mL for I hour and assayed by flow cytometry. Results shown in Figure 23 indicate that the free biotin at 10 g/mL did not displace significant amounts of the biotinylated epitope peptide. Numerical data for the inhibition assay is shown in Table 8.

This epitope mapping example demonstrates the useful application of the instant invention to the area of combinatorial screening. The peptide carrying the epitope for the mouse monoclonal antibody screened in this example was clearly identified in a set of nine peptides.
The identification was further shown to be specific by competitive inhibition with soluble epitope peptide. In addition, the stability of the avidin-biotin interaction for use with flow cytometry was demonstrated in an excess of free biotin.

MIF of Fm Bead Peptide Assayed Single Assayed Multiple plus GL-Biotin Single no peptide Multiple no peptide Peptide Assayed plus GL-Biotin w/free Biotin Bead Peptide MIF MIF
plus GL-Biotin Multiple w/ Biotin Multiple Analyte Simultaneous ToRCH Assay for Seroconversion.
This example demonstrates the utility of this invention in the screening of human serum for antibodies to infectious disease agents. Screening of serum for antibodies to certain infectious disease agents is often the only method available to determine if a patient has been, or is infected with the agent in question. For example, a common method of diagnosing HIV
infection is by detection of HIV specific antibodies in the serum. This phenomenon known as seroconversion is commonly employed for diagnosis of several important pathogenic infections.
io One of the most commonly employed assay panels of this type is the ToRCH
panel. ToRCH
assays detect both serum IgG and serum IgM responses to Toxoplasma gondii, Rubella virus, fvtomegalovirus, and Herpes Simplex Virus Types 1 and 2. The importance of this assay especially to the pregnant woman has been well documented as any one of these infectious agents is capable of crossing the placental barrier and entering the immunologically naive fetus.
These infectious agents can cause severe damage to the fetus and must be avoided. Currently , all ToRCH panel assays for antibodies specific to each of these pathogens is performed separately in a unique assay tube or microtiter well. This invention provides for a multiple analyte format that allows assay for either IgG or IgM antibodies specific for each of the five pathogens at the same time in the same tube with the same sample.

A ToRCH assay using flow cytometry has been developed by coupling purified antigens of T. gondii, Rubella, CMV and HSV Type 1 and Type 2 to five Differentially Fluorescent Microspheres (DFM). The specificity of the assay has been demonstrated by treating this bead set with human serum calibrators certified to be either positive or negative for all five agents.
After this treatment, the bead set was treated with either Goat anti-human IgG-Bodipy or Goat anti-human IgM-Bodipy used to develop the assay. In addition, a third calibrator with known levels of reactivity to each agent was assayed and the results reported.

Antibody labeling: Goat anti-human IgG and goat anti-human IgM (Cappel Division, Organon io Teknika, Durham, NC) were labeled with Bodipy FL-CASE (Molecular Probes, Inc., Eugene, OR) using methods described by the manufacturer of the Bodipy succinymidyl ester. Bodipy-labeled antibodies were stored in PBS containing 1 mg/mL BSA as stabilizer.

Antigen conjugation to microspheres: Five DFM (5.5 M carboxylate, Bangs Laboratories, Inc., is Carmel, IN, dyed by Emerald Diagnostics, Inc., Eugene, OR) were conjugated separately to the five ToRCH antigens (Viral Antigens, Inc.) with a two-step EDC coupling method (Pierce Chemicals, Rockford, IL) using sulfo-NHS to stabilize the amino-reactive intermediate. All antigens were dialyzed into PBS to remove any reactive amino groups such as sodium azide or glycine. The T. gondii preparation (Chemicon, Inc., Temecula, CA) was sonicated for 2 minutes 20 in PBS, 10 mM EDTA to lyse the tachyzoites. 20 L (8.4 million microspheres) of each bead type was activated for 20 minutes in a total volume of 100 gL containing 500 g of EDC and Sulfo-NHS in 50 mM sodium phosphate buffer, pH 7Ø Microspheres were washed twice with 200 .tL PBS, pH 7.4 using centrifugation at 13,400 x g for 30 seconds to harvest the microspheres. Activated and washed beads were suspended in 100 L of antigen at 0.05 to 0.15 25 mg/mL in PBS, pH 7.4. After 2 hours, the microspheres were blocked by addition of 100 L of 0.2 M glycine, 0.02% Tween 20 in PBS, pH 7.4 and incubated for an additional 30 minutes.
Antigen coated microspheres were washed twice with 200 L 0.02% Tween 20, 1 mg/mL BSA
in PBS, pH 7.4 (PBSTB). and stored in PBSTB at approximately 3,000,000 microspheres/mL as determined by hemacytometer count.

Rubella assay: Rubella antigen loaded microspheres were used to examine several parameters of the assay in a single analyte format prior to the performance of multiple analyte assays. 10 L
(30,000 microspheres) of Rubella antigen coated beads were reacted with 10 L
of a 1:10 dilution of four different Rubella calibrator sera (Consolidated Technologies, Inc., Oak Brook.

IL) and the mixture incubated for 1 hour. These sera were defined using a standard assay for the anti-Rubella IgG activity by the manufacturer of the calibrators. The units were defined as International Units/ mL or IU/mL. Beads were washed in PBSTB by centrifugation at 13,400 x g for 30 seconds and suspended in 40 L of a 10 g/mL solution of Bodipy-labeled anti-human IgG. This mixture was incubated for 1 hour, diluted to 300 tL in PBSTB and assayed using io flow cytometry. Negative controls included the microspheres with no serum treated with the Bodipy-labeled antibodies. In addition one calibrator serum containing 70 IU/mL of anti-Rubella IgG activity was titrated in a single analyte assay.

Multiple analyte assay for IgG and IgM activities: Equivalent amounts of each of the 5 antigen loaded microspheres was mixed to produce a ToRCH bead mixture. 10 .iL (30,000 microspheres) of the mixture was reacted with 10 L of a 1:400 dilution of ToRCH control or calibrator sera and incubated for 1 hour. The positive and negative ToRCH
control sera did not have defined units of activity. The ToRCH calibrator, however, did have defined levels of anti-TM
ToRCH IgG activities as defined by INX and DiaMedix diagnostic instruments.
These values were provided by the manufacturer for the lot of calibrator purchased. Beads were washed in PBSTB by centrifugation at 13,400 x g for 30 seconds and suspended in 20 L of a 40 g/mL
solution of Bodipy-labeled anti-human IgG or IgM'. This mixture was incubated for 1 hour, diluted to 300 gL in PBSTB and assayed using flow cytometry. Negative controls included the microspheres with no serum treatment and the microspheres treated with the ToRCH negative control serum. Both negative controls were developed with the Bodipy-labeled antibodies.

Results Rubella assay: Rubella coated DFM were reacted with 4 human serum calibrators containing known levels of IgG antibodies specific for Rubella virions defined by International units (IU/mL). The beads were washed and developed with goat anti-human IgG-Bodipy.
Results are shown in Table 9 and Figure 24. Increasing units of anti-Rubella activity were reflected in the Mean Intensity of Fluorescence (MIF) of F,,, (green channel). Luminex Units (LU) were defined as the MIF of F. for each data point minus the MIF of Fm for the negative control (no serum) multiplied by 0.1, and are included in Table 9.

Rubella calibrator titration: The human serum calibrator containing 70 IU/mL
of anti-Rubella IgG was serially diluted in PBSTB and assayed with the Rubella coated microspheres and Bodipy-labeled anti-human IgG. Results shown in Table 10 and Figure 25 show that, as io expected, the IgG antibodies specific for Rubella were titrated with dilution.

Multiple analyte ToRCH analysis for serum IgG and IgM: Each of the five distinct DFM coated with ToRCH antigens plus one DFM coated with human serum albumin (Miles, Inc., West Haven, CT) were mixed in equal volumes and 10 .tL (30,000 microspheres) of the mixture is reacted for 1 hour with triplicate, 20 gL aliquots of a 1:400 dilution of the ToRCH controls as well as the Low ToRCH calibrator. The calibrator from Blackhawk Systems, Inc.
contained known levels of each pathogen specific antibody as measured on other diagnostic machines.
After washing, one set of triplicates was developed with Bodipy-labeled anti-human IgG and another set with Bodipy-labeled anti-human IgM. Numerical results are shown in Tables 11 20 and 12. Results are presented graphically in Figures 26A and 26B. Included in the figures are standard deviation bars for the triplicate measurements. For both IgG and IgM
measurements, the ToRCH negative control serum (A96601, tubes #1-3) produced MIF of Fm similar to the negative control with no serum (tubes #10-12). The ToRCH positive control serum (A96602, tubes #4-6) demonstrated significant IgG activity to all five pathogens.
Conversely, the positive zs control serum had only slight IgM based reactivity to the five pathogens.
The known levels of anti-ToRCH IgG reactivities for the ToRCH Calibrator (A96500, tubes #7-9) were compared to the Luminex units of each IgG activity as determined by the multiple analyte analysis. Luminex units were defined by subtracting the negative control serum average MIF of Fm from the average MIF of F. for each antigen and multiplying by 0.1. The levels of the ToRCH calibrator were defined by the manufacturer as a factor of activity for each antigen above the limit of detection for that antigen on a specific diagnostic machine. These results are listed in Table 13.

A demonstrative ToRCH assay has been developed to simultaneously assay for serum s IgG or IgM specific for the five ToRCH pathogens in a single tube. Results of the assay indicate that it is specific for each pathogen and is as sensitive as currently available instrument based assays. The multiple analyte format provides a uniquely powerful technology for rapid and less expensive serum testing for seroconversion to ToRCH pathogens as well as other infectious agents diagnosed in this manner.

TABLE 9: Anti-Rubella calibration curve Calibrator IU/mL MIF of Fm LU/mL

WO 97/14028 - PCT/13S96/16198 , TABLE 10: Anti-Rubella calibrator titration 70 IU/mL Calibrator MIF of Fm Reciprocal of Dilution TABLE 11: IgG TORCH assay Calibrator MIF of Fm in Triplicate Tube # (1:400) Toxo. Rubella CMV HSV I HSV II HSA

No Serum 21 7 15 18 13 22 11 No Serum 23 8 11 15 19 19 12 No Serum 21 5 12 12 16 23 Calibrator Average MIF of Fm (1:400) Toxo. Rubella CMV HSV I HSV II HSA

No Serum 22 7 13 15 16 21 TABLE 12: IgM TORCH assay Calibrator MIF of Fm in Triplicate Tube # (1:400) Toxo. Rubella CMV HSV I HSV II HSA

No Serum 40 9 18 17 21 21 11 No Serum 36 8 20 17 19 16 12 No Serum 38 8 14 17 19 18 Calibrator Average MIF of Fm (1:400) Toxo. Rubella CMV HSV I HSV II HSA

No Serum 38 8 17 17 20 18 TABLE 13: Comparison of known levels of anti-ToRCH IgG for the ToRCH
calibrator from Blackhawk BioSystems with Luminex Units T.gondii Rubella CMV HSV I HSV 2 Diagnostic Machine used INX INX INX Diamedix DiaMedix Factor above Limit of Detection 1.7 x 2.7 x 1.7 x 2.5 x 1.1 x Units of Activity 11.3 IU/mL 26.9 IU/mL 24.5 IU/mL 50 EU/mL 22 EU/mL
Luminex Units/mL 7.2 LU/mL 3.2 LU/mL 3.8 LU/mL 11.1 LU/mL 4.3 LU/mL

Simultaneous Assay of Dog Sera for Allergic IgE and Allergen-Specific IgG
This example demonstrates the screening of serum for IgE antibodies specific for allergens. Screening of serum for IgE antibodies specific to allergens is a viable option for allergy testing as compared with skin sensitivity testing. The instant invention provides for a format that can assay for either IgG or IgE responses to numerous allergens at the same time in the same tube with the same sample and is therefore a uniquely powerful method of screening.
An allergy assay has been developed including 16 grass allergens in a multiple analyte, simultaneous format. A panel of 16 grass allergens were attached to 16 Differentially io Fluorescent Microspheres (DFM) with one grass allergen being coated onto one unique member of the bead set. The allergen bead set was treated with diluted dog serum for 1 hour and treated with a solution of either Goat anti-Dog IgE or goat anti-dog IgG-FITC for an additional hour.
For the IgE assay, beads were washed clear of this antibody and the bead set treated with an affinity purified rabbit anti-goat IgG-FITC antibody as probe.

Results demonstrate a uniquely powerful method of serum screening for allergies that provides a true multiple analyte format, as well as sensitivity and specificity.

Allergen conjugation to microspheres: Sixteen DFM (5.5 M carboxylate) were conjugated separately to 16 soluble grass allergens (provided by Dr. Bill Mandy, BioMedical Services, Austin, TX) with a two-step EDC coupling method (Pierce Chemicals, Rockford, IL) using sulfo-NHS to stabilize the amino-reactive intermediate. All grass allergens were diluted 1:100 into PBS, pH 7.4. 20 L (8.4 million microspheres) of each bead type was activated for 20 minutes in a total volume of 100 p.L containing 500 g of EDC and Sulfo-NHS in 50 mM
sodium phosphate buffer, pH 7Ø Microspheres were washed twice with 100 L
PBS, pH 7.4 using centrifugation at 13,400 x g for 30 seconds to harvest the microspheres.
Activated, washed beads were suspended in 50 L of diluted allergen. After 2 hours, the microspheres were blocked by addition of 50 L of 0.2 M glycine, 0.02% Tween 20 in PBS, pH
7.4 and incubated for an additional 30 minutes. Protein coated microspheres were washed twice with 100 .tL 0.02% Tween 20, 1 mg/mL BSA in PBS, pH 7.4 (PBSTB). and stored in PBSTB at approximately 3,000,000 microspheres/mL as determined by hemacytometer count.

Multiplexed K-9 grass allergen IgE assay: Equivalent amounts of each of the 16 grass allergen s loaded microspheres was mixed to produce a bead mixture. 20 L (60,000 microspheres) of the mixture was reacted with 60 L of a 1:3 dilution of dog serum in PBSTB and the mixture incubated for 1 hour. Beads were washed in 200 L PBSTB by centrifugation at 13,400 x g for 30 seconds and suspended in 40 L of a 50 g/mL solution of anti-dog IgE
(provided by Dr.
Bill Mandy, BioMedical Services, Austin, TX). After incubation for 1 hour, beads were washed io in 200 L PBSTB by centrifugation at 13,400 x g for 30 seconds. Beads were then treated with 40 L of rabbit anti-goat IgG-FITC (Sigma, St. Louis, MO) at 20 g/mL. After one hour the bead mixture was diluted to 300 L in PBSTB and assayed using flow cytometry.
Negative controls included the microspheres with dog serum, without the goat anti-dog IgE and with the rabbit anti-goat IgG-FITC. A negative control of the bead set with no dog serum was also is included. Allergen specific dog IgE was determined by subtraction of the mean intensity of fluorescence (MIF) of the green channel (Fm) for the negative controls, for each grass allergen from the MIF of Fm for the tubes including the goat anti-dog IgE.

Simultaneous K-9 grass allergen IgG assay: Equivalent amounts of each of the 16 grass allergen 20 loaded microspheres was mixed to produce a bead mixture. 20 L (8.4 million microspheres) of the mixture was reacted with 20 L of a 1:10 dilution of dog serum in PBSTB
and the mixture incubated for 1 hour. Beads were washed in 200 p.L PBSTB by centrifugation at 13,400 x g for 30 seconds and suspended in 25 L of a 50 .tg/mL solution of goat anti-dog IgG-FITC. After one hour the bead mixture was diluted to 300 L in PBSTB and assayed using flow cytometry.
25 Negative controls included the microspheres with no dog serum and with the goat anti-dog IgG-FITC. Allergen specific dog IgG was determined by subtraction of the mean intensity of fluorescence (MIF) of the green channel (Fm) for the negative control for each grass allergen from the MIF of F. for the tubes including dog serum.

Results Multiple anal tag anti-grass allergen IgG assay: Grass allergen coated DFM
were reacted with 6 dog sera provided by BioMedical Services, Austin, TX that had been characterized by ELISA for anti-grass allergen IgE. The IgG response to these grass allergens was not measured s by BioMedical Services. The beads were washed and developed with goat anti-dog IgG-FITC.
Results are shown in Figure 27. The MIF of Fm in the absence of dog serum was subtracted from the MIF of Fm for each bead with each dog serum. Two dogs, A96324 and demonstrated relatively high IgG reactivity to most of the grass allergens.
Two dogs, A96325 and A96317 demonstrated relatively medium IgG reactivity to most of the grass allergens. Two io dogs, A96319 and A96323 demonstrated relatively low IgG reactivity to most of the grass allergens.

Multiple analvte dog anti-grass allergen IgE w: Grass allergen coated DFM were reacted with 6 dog sera provided by BioMedical Services, Austin, TX that had been characterized by is ELISA for anti-grass allergen IgE. The beads were washed and treated with goat anti-dog IgE
for I hour. The assay was developed with rabbit anti-goat IgG-FITC. Results are shown in Figure 28. The MIF of Fm in the absence of dog serum was subtracted from the MIF of Fm for each bead with each dog serum. Two dogs, A96325 and A96326 demonstrated relatively low reactivity to most of the grass allergens with the exception of Wheat grass and several others for 20 A96326. These results agree with the ELISA results provided by BioMedical Services. A96325 was negative for 11 grass allergens (only ones tested) and A96326 was negative for the same I 1 grass allergens except for a "Borderline" result in ELISA against a mixture of Wheat and Quack grass (due to the non-multiplexed format of ELISA assays, allergens are often mixed to increase the throughput levels). The other four dog sera demonstrated medium to high IgE responses to 25 several of the grass allergens. Although agreement between ELISA and flow cytometry assay results was not absolute, the two assays followed the same trends. Dogs with IgE reactivity to grass allergens were detected by both assays.

Comparison of multiple anal ty e IgG and IgE results: The IgG and IgE anti-grass allergen 3o response to each of the 16 allergens was compared by graphing. Figures 29-34 demonstrate that there was no correlation between IgG and IgE response to grass allergens in the six dogs. Some dogs were low responders for both IgE and IgG, some were reactive with both immunoglobulin subclasses, and some demonstrated IgE reactivity in a low background of IgG
specific for the grass allergens. Examination of the IgG reactivity in a serum could identify those sera in which the IgE reactivity could be masked by the high IgG reactivity.

A demonstrative assay for serum IgG or IgE activity to 16 grass allergens has been developed that allows simultaneous assay of all 16 allergens at the same time in the same tube using the same sample. Results with 6 dog sera suggested that IgE anti-grass allergen activity as io determined by ELISA was in general agreement with results provided using flow cytometry. In addition, the ease of determination for IgG anti-grass allergen activity in the six dogs was demonstrated.

A Simultaneous Immunometric Assay For Human Chorionic Gonadotropin and Alpha-Fetoprotein This example illustrates the determination of multiple analyte levels in a liquid sample simultaneously by immunometric or capture-sandwich assay. The use of capture-sandwich assays to accurately determine analyte levels in liquid solutions is a commonly used format for many analyte assays. The technique is especially useful for those analytes present in low quantities as the first step serves to capture and thus concentrate the analyte. The uniqueness of this assay is the multiple analyte format allowing the simultaneous determination of two distinct serum proteins at the same time in the same tube from the same serum sample.

Human chorionic gonadotropin (hCG), a gonadotropic hormone secreted by the placenta, is the primary hormonal marker utilized for pregnancy testing. hCG is elevated both in urine and serum during pregnancy. Alpha fetoprotein (AFP) is the fetal cell equivalent to human serum albumin. AFP is elevated in pregnancy and in certain types of malignancies. Many clinical fertility or pregnancy test panels include immunometric assays for these two serum proteins. Immunometric or capture-sandwich assays for hCG and AFP were developed separately and then combined in a multiple analyte format.

The hCG assay was developed by examining several antibody pairs for their ability to capture and quantitate hCG levels in solution. First, a monoclonal antibody was coupled through carbodiimide linkage to a carboxylate substituted Differentially Fluorescent Microsphere (DFM). Next, a polyclonal, affinity purified antibody was Bodipy-labeled and s used to probe DFM captured hormone. Once this assay was adjusted to include physiological sensitive ranges, the process was repeated for AFP. Cross-reactivity of the two assays was examined to demonstrate that the two assays would not interfere. The assays were then performed simultaneously. Commercially available serum calibrators were used to demonstrate that both assays were sensitive in clinically relevant ranges and an unknown was include to io demonstrate how the two assays work simultaneously.

Antibody labeling: The two affinity purified polyclonal anti-hCG (AB633) and anti-AFP
(M20077) antibodies (Chemicon, Inc., Temecula, CA and Medix Division, Genzyme, San Carlos, CA) were labeled with Bodipy FL-CASE (Molecular Probes, Inc., Eugene, OR) using 15 methods described by the manufacturer of the Bodipy succinymidyl ester. The resulting Bodipy-labeled antibodies were stored in PBS containing 1 mg/mL BSA as stabilizer.

Antibody conjugation to microspheres: Monoclonal anti-hCG (MAB602) and anti-AFP
(S10473) capture antibodies were conjugated to microspheres with a two-step EDC coupling 20 method (Pierce Chemicals, Rockford, IL) using sulfo-NHS to stabilize the amino-reactive intermediate. 20 }.tL ( 8.4 million microspheres) of each DFM was activated for 20 minutes in a total volume of 100 L containing 500 p.g of EDC and Sulfo-NHS in 50 mM sodium phosphate buffer, pH 7Ø Microspheres were washed twice with 200 L PBS, pH 7.4 using centrifugation at 13,400 x g for 30 seconds to harvest the microspheres. Washed, activated beads were 25 suspended in 50 p.L of a 0.05 mg/mL solution of antibody in PBS, pH 7.4.
After 2 hours, microspheres were blocked by addition of 50 L of 0.5 mg/mL BSA, 0.02% Tween 20 in PBS, pH 7.4 and incubated for an additional 30 minutes. Protein coated microspheres were washed twice with 200 L 0.02% Tween 20, 1 mg/mL BSA in PBS, pH 7.4 (PBSTB) and stored in PBSTB at approximately 3,000,000 microspheres/mL. Microsphere concentrations were 3o determined using a hemacytometer.

Antibody pairs analysis of hormone capture assay. Capture assay antibody pairs were screened by coupling potential capture antibodies to microspheres and assaying them using all potential combinations of capture antibody-bead/ Bodipy-labeled probe antibody. Assays were performed using 10 pL of capture antibody microspheres (approximately 30,000) plus 20 pL
of antigen s solution at 10 pg/mL in PBSTB for a 1 hour incubation. Beads were washed in PBSTB by centrifugation at 13,400 x g for 30 seconds and suspended in 20 pL of a 25 pg/mL solution of Bodipy-labeled probe antibody. This mixture was incubated for 1 hour, diluted to 300 pL in PBSTB and assayed using flow cytometry.

io Antigen titration assay Once an antibody pair was chosen for use, the pair was analyzed for sensitivity and limit of detection by titration of antigen. Assays were performed using 10 pL of capture antibody microspheres plus 20 pL of antigen dilutions in PBSTB for a 1 hour incubation. Beads were washed in 200 pL PBSTB by centrifugation at 13,400 x g for 30 seconds and suspended in 20 pL of a 25 pg/mL solution of Bodipy-labeled probe antibody.
1s This mixture was incubated for 1 hour, diluted to 300 gL in PBSTB and assayed using flow cytometry.

Cross-reactivity analysis: To examine the possibility of cross-reactivity, 10 pL of MAB 602 anti-hCG capture beads (5,000 microspheres) were treated with 20 pL dilutions of hCG or AFP.
20 After 1 hour the beads were washed in 200 pL PBSTB by centrifugation at 13,400 x g for 30 seconds and suspended in 20 pL of either Bodipy-labeled anti-hCG or Bodipy-labeled anti-AFP
at 25 pg/mL. Conversely, 10 pL of S-10473 anti-AFP capture beads (5,000 microspheres) were treated with 20 pL dilutions of hCG or AFP. After 1 hour, beads were washed in 200 [LL
PBSTB by centrifugation at 13,400 x g for 30 seconds and suspended in 20 pL of either Bodipy-25 labeled anti-hCG or Bodipy-labeled anti-AFP at 25 pg/mL. Mixtures were incubated for 1 hour, diluted to 300 pL in PBSTB and assayed using flow cytometry.

Washed vs. no-wash assay format: An AFP/hCG capture antibody bead mixture was made by mixing equal amounts of the two bead types. In duplicate, 10 pL of this bead mixture (10,000 30 microspheres) was mixed with 20 pL dilutions of AFP/ hCG and incubated for 1 hour. One set of beads were washed in PBSTB by centrifugation at 13,400 x g for 30 seconds and suspended in 20 pL of a mixture of Bodipy-labeled anti-hCG and anti-AFP both at 25 g/mL. This mixture was incubated for 1 hour, diluted to 300 L in PBSTB and assayed using flow cytometry. The second set of beads were treated directly with 20 L of a mixture of Bodipy-labeled anti-hCG and anti-AFP both at 25 p.g/mL. This mixture representing a homogenous (no-wash) assay was also incubated for 1 hour, diluted to 300 L in PBSTB and assayed using flow cytometry.

Multiple analyte assay: Once the AFP and hCG antibody pairs were shown not to cross-react io and were adjusted to provide clinically relevant ranges of sensitivity in a homogenous assay, the assays were performed simultaneously using commercially available serum calibrators as the source of AFP and hCG antigens. Equivalent amounts of each of the two capture antibody loaded microspheres was mixed to produce an AFP/hCG capture mixture. In triplicate, 10 L of this bead mixture (5,000 of each microsphere) was mixed with 20 L of three serum calibrators is (high, medium and low) containing known levels of AFP and hCG and incubated for 1 hour.
Mixtures were treated directly with 20 L of a blend of Bodipy-labeled anti-hCG and anti-AFP
both at 25 g/mL. Mixtures were incubated for 1 hour, diluted to 300 L in PBSTB and assayed by flow cytometry.

20 Results Antibody pair analysis for hCG capture assay: For hCG antibody pair analysis, five capture antibody/microspheres were prepared and the identical five antibodies were Bodipy-labeled to serve as probes. Three of the antibodies were specific for the alpha sub-unit of hCG and two for the beta sub-unit. The three anti-alpha sub-unit antibody/microspheres were assayed for utility 25 with the two Bodipy-labeled anti-beta hCG antibodies. Conversely, the two anti-beta sub-unit antibody/microspheres were assayed for utility with the three Bodipy-labeled anti-alpha hCG
antibodies. Results of this screen are shown in Table 14 and Figure 35. The 12 combinations of antibodies are shown with (odd numbers) and without (even numbers) hCG at 20 g/ml,. It is apparent that the first two antibody pairs, #1 and #3 demonstrated the highest mean intensity 30 of fluorescence (MIF) of the F,,, (green channel). Further examination of these two pairs led to the decision to chose the #3 pair of MAB 602 for capture antibody and AB633-Bodipy as probe antibody for the hCG capture/sandwich assay.

Antigen titration: The MAB 602/AB633 anti-hCG capture system was assayed by hCG titration s to determine if the level of sensitivity required for clinical assay was achievable. A limit of detection of at least 1 ng hCG/mL was the target as this was the level of hCG
in the low serum calibrator to be used later in this project. The results of this antigen titration is shown in Table 15 and Figure 36. The limit of detection was between 20 and 200 pg/mL. This revealed that the MAB602/AB633 anti-hCG antibody pair was sufficiently sensitive for hCG
analysis.
io Included in this analysis was MIF of F,,, measurements from counting of 100 or 1000 microspheres. Results were similar. A similar analysis of antibody pairs and antigen titration for AFP identified an AFP pair that could be further developed.

Cross-reactivity assay: The MAB 602/AB633 anti-hCG capture system and S-is anti-AFP capture system were examined for cross reactivity by assaying each capture bead with each antigen and Bodipy-labeled antibody. Results are shown in Table 16 and Figures 37A and 37B. No significant cross-reactivity between the hCG and AFP capture systems was detected.
No-wash vs. washed assay format: The hCG and AFP assays were performed simultaneously 20 and examined for the limit of quantitation or dynamic range in both a washed format and no wash or homogenous format. Result of these antigen titrations are shown in Table 17 and Figures 38A and 38B. Results indicated that the homogenous format provided sufficient dynamic range for the purposes of clinical relevance.

25 Multiple analyte hCG/AFP assay: The two assays were performed simultaneously using serum calibrators of known hCG and AFP levels to generate a standard curve. For each standard curve one serum of unknown hCG and AFP level was included to demonstrate how the assay would determine the level of hCG and AFP in the serum.

The Randox Tri-level calibrators consisted of three serum samples with high, medium and low levels of hCG and AFP documented in mU or U/mL for hCG and AFP
respectively.
These calibrators are used in at least 12 different diagnostic instruments including those of Abbott (Abbott Park, IL), bioMerieux (St. Louis, MO), Ciba Corning (Medfield, MA), s Diagnostics Products (Los Angeles, CA), Kodak (Rochester, NY), Syva (San Jose, CA), Tosoh (Atlanta, GA) and Wallac (Gaithersburg, MD). Literature with the Randox Tri-Level control listed the units of each known analyte as defined by each diagnostic machine.
We calculated the average of the hCG mU/mL and AFP U/mL for the three calibrators. In the case of the hCG, the low and medium calibrators contained 22.8 and 26.4 mU/mL which were extremely close to considering the distance to the high calibrator (436 mU/mL). For this reason, we included a 1:2 dilution of the high range calibrator into hCG/AFP certified negative serum to produce a fourth level serum calibrator termed Level 3D. Calibrator 3D was only used in construction of the hCG
standard curve so each of the assays was effectively defined by three point calibration.

15 Table 18 shows the results of this multiple analyte assay. The assay was performed in triplicate and the average MIF of F. computed for graphing. Coefficients of variation (CV) for the triplicates were consistently less than 10% are shown. Also included in the table are the number of microspheres correctly identified by the flow cytometry out of the 400 counted per tube. Of the 400 beads counted the expected ratio of MAB 602 containing 60/40 beads to S-20 10473 containing 40/60 beads was 1:1. Therefore of the 200 beads expected, this was the number of beads correctly identified and used to compute the MIF of F, for that data point.
Figures 39A and 39B graphically represent the data of Table 18. For both hCG
and AFP a plot of the MIF vs. the log of antigen concentration produced a line that was best fit using 25 a third level polynomial equation. The fit for the hCG curve provided an R2 of 1.0 and for AFP
an R2 of 0.9999 was achieved. Using the polynomial equation, the concentration of the unknowns was computed. Results of these analyses are seen in Table 18. The unknown serum contained 218.55 6.56 mU/mL of hCG and 39.59 1.19 U/mL of AFP.

A
demonstrative immunometric assay for hCG and AFP in serum has been developed.
Assays were first developed as single analyte or single bead assays, and optimized with regards to sensitivity, limit of quantitation and cross-reactivity. The assays were then combined to quantitatively determine multiple analyte levels in a liquid solution in the same tube from the s same sample at the same time. Results, using commercially available calibrator sera, has proven that this invention is effective for this type of quantitative assay.

Sample Description hCG Conc. ( g/mL) MIF of Fm 1 A l-Beads + B I Ab-BD with hCG 20.0 8790 2 A 1-Beads + B 1 Ab-BD with no hCG 0.0 108 3 A2-Beads + B I Ab-BD with hCG 20.0 9441 4 A2-Beads + BI Ab-BD with no hCG 0.0 163 A3-Beads + B 1 Ab-BD with hCG 20.0 3150 6 A3-Beads + B1 Ab-BD with no hCG 0.0 2984 7 A I -Beads + B2 Ab-BD with hCG 20.0 2287 8 A I -Beads + B2 Ab-BD with no hCG 0.0 37 9 A2-Beads + B2 Ab-BD with hCG 20.0 1232 A2-Beads + B2 Ab-BD with no hCG 0.0 42 11 -A3-Beads + B2 Ab-BD with hCG 20.0 566 12 A3-Beads + B2 Ab-BD with no hCG 0.0 560 13 B1-Beads + Al Ab-BD with hCG 20.0 70 14 BI-Beads + Al Ab-BD with no hCG 0.0 23 B2-Beads +A I Ab-BD with hCG 20.0 346 16 B2-Beads + Al Ab-BD with no hCG 0.0 20 17 B 1-Beads + A2 Ab-BD with hCG 20.0 107 18 B1-Beads + A2 Ab-BD with no hCG 0.0 33 19 B2-Beads + A2 Ab-BD with hCG 20.0 886 B2-Beads + A2 Ab-BD with no hCG 0.0 56 21 BI-Beads + A3 Ab-BD with hCG 20.0 105 22 B1-Beads + A3 Ab-BD with no hCG 0.0 196 23 B2-Beads + A3 Ab-BD with hCG 20.0 143 24 B2-Beads + A3 Ab-BD with no hCG 0.0 609 Sample hCG Conc. (ng/mL) MIF of Fm (1000 Beads) MIF of Fm (1000 Beads) 6 0.2 258 254 7 0.02 120 147 8 0.002 122 121 9 0.0002 122 149 MAB602 BEADS - Anti-hCG
Samp. Antigen hCG AFP hCG AFP
ng/mL anti-hCG anti-hCG anti-AFP anti-AFP
1 1000.0 792 53 47 52 2 100.0 761 47 48 48 3 10.0 530 47 47 48 4 1.0 104 47 48 48 0.1 55 52 49 48 6 0.0 48 71 72 48 M20077 BEADS - Anti-AFP
Samp. Antigen hCG AFP hCG AFP
ng/mL anti-hCG anti-hCG anti-AFP anti-AFP
1 1000.0 99 57 78 348 2 100.0 54 75 44 356 3 10.0 44 44 45 103 4 1.0 51 50 44 98 5 0.1 42 75 49 44 6 0.0 43 61 45 45 TABLE

AFP hCG
Sample AFP No Wash Washed hCG No Wash Washed No. ng/mL ng/mL

8 8_ 190 205 16 2440 2528 12 0.5 24 25 1 330 359 13 0.25 17 24 0.5 166 175 hCG capture system AFP capture system Tube Descript. hCG AFP MIF of MIF MIF Beads MIF of MIF MIF Beads No. mU/mL U/mL FLI AVG CV% IDed FL1 AVG CV% IDed 1 Level l h< ' 23 114 74 98 2 Level1 22.8 10.7 27 25.67 7% 130 83 78.00 5% 80 3 Level l 27 140 77 72 4 Leve12 32 93 351 58 Leve12 26.4 53.8 34 i 31.67 W 6% 85 365 r362.67 61 6 Level 2 29 f.:. 94 372 ::....:...... 65 .
7 Level 3D 268 92 535 56 8 Level 3D 218 111.5 276 271.67 1% 101 562 1552.00 2% V 69 =: ,; ........ 61 9 Level 3D 271 96 559 Leve13 :.....:. , ;aa 631 106 1109 :<> 46 I1 Leve13 436 223 601 624.00 3% 99 994 1061.00 5% 38 12 Level 3 640 OJIMIS
JI
``': 97 1080 40 13 Negative 8 99 11 104 ------ - -----14 Negative 10 2 7 7.33 6% 111 13 12.33 8% 106 Negative .... 7 119 13 s .... 95 16 Unknown 270 140 268 67 17 Unknown 218.55 39.59 264 272.33 141 274 276.00 3% 81 18 Unknown 283 113 286 ~::; : 84 Multiplexed Beadset Standard Curve Using an Inhibition Assay This example provides a demonstration of the measurement of ligand-ligate reactions using a multiplexed beadset standard curve. Commonly for ligand-ligate reactions quantitation, known amounts of the ligand or ligate are introduced to the reaction leading to the production of a standard curve. Values for unknown samples are compared to the standard curve and quantified. The true multiple assay capability of this invention allows for an additional type of standard to be utilized. A multiplexed beadset standard curve for measuring analyte concentration is created by using several Differentially Fluorescent Microspheres (DFM) coated with either 1) different amounts of ligand (antigen), or 2) different amounts of ligate (antibody), io or 3) different ligates possessing different avidities for the ligand (different monoclonal antibodies). We have demonstrated an example of the first type of multiple analyte standard curve by developing a competitive inhibition assay for human IgG.

Four DFM were coated with human IgG at four different concentrations. When probed 1s with goat anti-human IgG-Bodipy the Mean Intensity of Fluorescence (MIF) of F,n (green channel) for each bead subset was different. The MIF of Fm correlated with the amount of hlgG
used to coat the beads in each subset. If soluble hIgG was mixed with the reaction in a competitive manner the MIF of Fm was reduced for each bead as less of the probe antibody was bound to the beads. In a normal standard curve, the signal (MIF of Fm) is plotted against the 20 concentration of the inhibitor. For the multiplexed beadset standard curve, the slope of the MIF
of Fm across the beads within a subset is plotted against the concentration of inhibitor.
Comparison of the two types of standard curves revealed them to be of equivalent value for prediction of an unknown amount of inhibitor.

25 Human IgG conjugation to microspheres: Four DFM (5.5 M carboxylate, Bangs Laboratories, Inc., Carmel, IN, dyed by Emerald Diagnostics, Inc., Eugene, OR) were conjugated separately to 4 different concentrations of hIgG (Cappel Division, Organon Teknika, Durham, NC) with a two-step EDC coupling method (Pierce Chemicals, Rockford, IL) using sulfo-NHS
to stabilize the amino-reactive intermediate. 20 L (8.4 million microspheres) of each bead type was 3o activated for 20 minutes in a total volume of 100 p.L containing 500 g of EDC and Sulfo-NHS

in 50 mM sodium phosphate buffer, pH 7Ø The microspheres were washed twice with 200 L
PBS, pH 7.4 using centrifugation at 13,400 x g for 30 seconds to harvest the microspheres.
Activated, washed beads were suspended in 50 L of hlgG at 50, 10, 5, and 1 g/mL in PBS, pH 7.4. After 2 hours, the microspheres were blocked by addition of 50 gL of 0.2 M glycine, s 0.02% Tween 20 in PBS, pH 7.4 and incubated for an additional 30 minutes.
Protein coated microspheres were washed twice with 200 L 0.02% Tween 20, 1 mg/mL BSA in PBS, pH 7.4 (PBSTB). and stored in PBSTB at approximately 3,000,000 microspheres/mL as determined by hemacytometer count.

so Antibody labeling: Goat anti-human IgG (Cappel Division, Organon Teknika, Durham, NC) was labeled with Bodipy FL-CASE (Molecular Probes, Inc., Eugene, OR) using methods described by the manufacturer of the Bodipy succinymidyl ester. The resulting Bodipy-labeled antibody was stored in PBS containing 1 mg/mL BSA as stabilizer.

is Multiplexed beadset standard curve: Equivalent amounts of each of the 4 differentially loaded IgG microspheres was mixed to produce a bead mixture. 10 L of the goat anti-hIgG-Bodipy at 25 g/mL in PBSTB was mixed with 10 L of a dilution of hIgG in PBSTB.
Immediately 10 gL (30,000 microspheres) of the bead mixture was added and the mixture incubated at room temperature for 30 minutes. Beads were diluted to 300 L in PBSTB and assayed using flow 20 cytometry. A negative control included the microspheres with the goat anti-hIgG-Bodipy with no inhibitor (hIgG). Each bead subset was assigned the value of a consecutive integer (i.e. the bead subset coupled with the lowest concentration of IgG was given a value of 1, the next highest concentration was given a value of 2, etcetera) and those numbers plotted against the MIF of each bead subset at each concentration of inhibitor tested. The slopes (designated here 25 as inter-bead subset slopes) were computed using linear regression analysis. The inter-subset slopes were then plotted against the concentration of inhibitor using a logarithmic scale for the concentration of inhibitor. Results were plotted as the slope of the MIF of F,,, across the bead set against the log of hIgG concentration. Curve fitting was performed using a power function trendline and the R2 correlation was reported. For a perfect fit, R2=1Ø

Common standard curve: Using the data from the assay described above, a common standard curve was constructed to compare results with the multiple analyte standard curve. Data from the DFM coated at 50 g/mL hIgG was utilized to produce a five-point standard curve by plotting the MIF of F. against the log of hIgG concentration. Curve fitting was performed using a power function trendline and the R2 correlation was reported.
Results Multiplexed beadset standard curve for a competitive inhibition assay Four differentially loaded IgG microspheres were utilized in a multiple beadset competitive inhibition assay for hIgG at io five different concentrations of soluble inhibitor (hIgG). Results of the assay are shown in Table 19. The inhibition pattern on each bead is plotted in Figure 40. The inter-bead subset slopes are plotted against the log concentration of inhibitor in Figure 41. A
Power Trendline in Excel was used to produce the R2 of 0.9933.

Common standard curve using one bead of the multiple analyte assay: Data from the 50 g/mL
hIgG bead was utilized to produce a five-point standard curve by plotting the MIF of F,,, against the log of hIgG concentration. Results are shown in Figure 42. Curve fitting was performed using a Power function trendline and R2 = 0.9942.

A novel type of standard curve for ligand-ligate measurement was demonstrated.
Results suggested that the multiplexed beadset standard curve was of similar utility as the common multi-point standard curve in quantitation of unknown samples. Advantages of the multiplexed beadset standard curve include the inclusion of the standard curve microspheres in each point of a multiplexed beadset assay, and the extension of an assay's dynamic range.
This may be 2s achieved by increasing the concentration range of ligand or ligate on the microspheres or by increasing the range of avidities for ligand on a series of microspheres.

Samp Inhibitor Bead 1 Bead 2 Bead 3 Bead 4 SLOPE
Conc ( g/mL) 1.0 g/mL IgG 5 pg/mL IgG 10 gg/mL IgG 50 g/mL IgG
1 100 14 77 108 288 85.3 2 50 21 100 162 428 128.3 3 25 40 166 267 844 251.3 4 12.5 110 463 747 1467 435.5 6.25 257 1226 1629 2316 658 6 0 134 793 1432 2217 688.8 Nucleic Acid Measurement The power and sensitivity of PCR has prompted its application to a wide variety of 5 analytical problems in which detection of DNA or RNA sequences is required.
One major difficulty with the PCR technique is the cumbersome nature of the methods of measuring the reaction's products - amplified DNA.

A major advance in this area is here. That advance employs a flow cytometric bead-1o based hybridization assay which permits the extremely rapid and accurate detection of genetic sequences of interest. In a preferred embodiment of that invention, a bead to which a nucleic acid segment of interest has been coupled is provided. A PCR product of interest (or any other DNA or cDNA segment) is detected by virtue of its ability to competitively inhibit hybridization between the nucleic acid segment on the bead and a complementary fluorescent nucleic acid is probe. The method is so sensitive and precise as to allow the detection of single point mutations in the PCR product or nucleic acid of interest. Although that method in itself provides a pivotal advance in the art of analyzing PCR reaction products, the further discovery of methods of multiplexing such an analysis, compounds the method's power and versatility to allow simultaneously analysis of a number of nucleic acid products or a number of sequences within a 20 single product in a single sample.

The multiplexed DNA analysis method described here can be applied to detect any PCR
product or other DNA of interest for specific polymorphisms or mutations or for levels of expression, e.g. mRNA. With the multiplexed techniques provided by the instant invention.
individuals can be screened for the presence of histocompatibility alleles associated with s susceptibility to diseases, mutations associated with genetic diseases, autoimmune diseases, or mutations of oncogenes associated with neoplasia or risk of neoplasia. The analysis of DNA
sequences occurs generally as follows:

1. A beadset containing subsets of beads coupled to nucleic acid sequences of interest is io prepared by coupling a unique synthetic or purified DNA sequence to the beads within each subset.

2. Fluorescent probes complementary to the DNA coupled to each bead subset are prepared.
Methods known in the art, e.g., as described in U.S. Patent No. 5.403,711, issued April 4.
1995 which may be referred to for further details or other methods may be used to fluo-1; rescently label the DNA. Since each probe will bind optimally only to its complementary DNA-containing subset, under the conditions of the assay, the fluorescent probes may be added to the subsets before or after the subsets are pooled, and before or after addition of the DNA test sample(s) of interest.

3. Tissue, fluid or other material to be analyzed is obtained, and DNA is purified and/or 20 amplified with PCR as necessary to generate the DNA products to be tested.

4. The DNA samples of interest are then mixed with the pooled beadset under suitable conditions to allow competitive hybridization between the fluorescent probes and the DNA
of interest.

5. The beadset is then analyzed by flow cytometry to determine the reactivity of each bead 25 subset with the DNA sample(s). If the test sample contains a DNA sequence complementary to the DNA of a given bead subset then that subset will exhibit a decreased Fm value relative to the F. value of beads to which a control DNA has been added. A
computer executed method in accordance with the current invention can determine the subset from which each bead is derived, and therefore, the identity of the DNA
sequence 30 on the bead and any change in Fm.

Detection of Foreign DNA
The methods of the present invention find wide utility in the detection of foreign DNA's in, for example, diagnostic assays. Although the DNA segment to be analyzed can be any DNA
sequence, in accordance with this embodiment the selected segment will be a DNA segment of a pathogenic organism such as, but not limited to, bacterial, viral, fungal, mycoplasmal, rickettsial, chlamydial, or protozoal pathogens. The procedure has particular value in detecting infection by pathogens that are latent in the host, found in small amounts, do not induce inflammatory or immune responses, or are difficult or cumbersome to cultivate in the laboratory.
The multiplexed DNA detection method of the present invention is likely to find particular io utility as a diagnostic assay for analysis of a sample from a patient having clinical symptoms known to be caused by a variety of organisms using a beadset designed to detect DNAs from the variety of organisms known to cause such symptoms to determine which of such organisms is responsible for the symptoms. DNA would be extracted from tissue, fluid or other sources and analyzed as described above.

Analysis of Genetic Polymorphisms The invention may also be used to measure a variety of genetic polymorphisms in a target DNA of interest. For example, there are several genes in the MHC and many are polymorphic. There are at least two applications in which determination of the alleles at each position of the MHC is of critical importance. The first is the determination of haplotype for transplantation, and the second is determination of haplotype as indicator of susceptibility to disease. See Gross et al., "The Major Histocompatibility Complex-Specific Prolongation of Murine Skin and Cardiac Allograft Survival After In Vivo Depletion of VR+ T
Cells," J. Exp.
Med., 177, 35-44 (1993). The MHC complex contains two kinds of polymorphic molecules, Class I genes, HLA A, B and D which have 41, 61 and 18 known alleles and Class 10 genes, HLA-DRI,3,4,5 HLA-DQAI and BI HLA-DP, DPAI, DPB1, also with many alleles. Each human can have up to 6 co-dominant Class I genes and 12 co-dominant Class 10 genes.

In the case of transplantation, the closer the match between the donor and recipient the greater the chance of transplant acceptance. A multiplexed assay in accordance with the invention may be employed to perform tissue typing quickly and accurately to identify suitable matches for transplantation.

In the situation of disease association, it has been found that individuals bearing certain s alleles are more prone to some diseases than the remainder of the population. The frequency of alleles of the MHC genes is not equal, and sets of alleles are frequently found (linkage disequilibrium) so that the identification of the exact set of alleles associated with many diseases is feasible. As one 'example, insulin-dependent diabetes mellitus (IDDM) is associated with certain HLA-DQ alleles. The number of alleles of DQ in the population is modest and genetic io typing by PCR amplification and hybridization with allele specific probes has been shown to be practical. See Saiki et al., "Genetic Analysis of Amplified DNA with Immobilized Sequence-Specific Oligonucleotide Probes," Proc. Natl. Acad. Sci. U.S.A., 86, 6230-6234 (1989).

For an assay of MHC in accordance with the invention, DNA is obtained from blood or 15 other extractable source, and amplified with primers specific for the MHC
genes under analysis, for example, HLA-DQA. For a full genotyping of the MHC, several samples of DNA
would be amplified with different sets of primers to accommodate the large number of loci and the high degree of polymorphism. The PCR products are then screened for specific alleles using beadsets and fluorescent probes as described above.
Mutation Analysis of Selected Genes: Screening Procedures There are several methodologies for determining and comparing DNA sequences in order to detect mutations which are associated with disease or neoplasia. When adapted to a bead-based, multiplexed format in accordance with the current invention, hybridization analysis allows for the rapid screening of multiple genetic loci for multiple wild type and mutant sequences.

In a preferred embodiment of the invention, a given genetic locus, or multiple loci, can be simultaneously screened for the presence of wild type or mutant sequences.
In the same 3o analysis, multiple known mutations can be distinguished from each other and from the wild type sequence and uncharacterized mutations. In addition, the homozygosity or heterozygosity of known sequences can be determined.

A general approach for detecting a DNA mutation in accordance with this aspect of the invention is as follows. In a first step, a suitable probe for detecting a mutation of interest is selected. In an illustrative embodiment, selected oligonucleotides, representing wild-type and mutant sequences, from a region of a gene known to contain a mutation are prepared. Such oligonucleotides are coupled to microspheres by techniques known in the art, (e.g., carbodiimide coupling, or other means) to produce individual aliquots of beads having known oligonucleotides io coupled thereto. The oligonucleotides must be a sufficient length to allow specific hybridization in the assay, e.g., generally between about 10 and 50 nucleotides, more preferably between about 20 and 30 nucleotides in length. In a preferred embodiment, a saturating amount of the oligonucleotideis bound to the bead. Fluorescent oligonucleotides, complementary to all or part of the sequences attached to each bead, are also prepared.

Next, PCR primers are selected to amplify that region of the test DNA
corresponding to the selected probe, which are then used to amplify the particular region of DNA in the sample that contains the sequence corresponding to the oligonucleotide coupled to the beads. Either double stranded or single stranded PCR techniques may be used. If double stranded product is produced, the amplified PCR product is made single stranded by heating to a sufficient temperature to and for a sufficient time to denature the DNA (e.g., for about 1 to about 5 minutes at about 90-95 C in 2.3X' SSC hybridization buffer). The mixture is cooled, and the beads are added and incubated with the PCR product under conditions suitable to allow hybridization to occur between the oligonucleotide on the beads and the PCR
product (e.g., at room temperature for about 10 minutes). The fluorescent DNA probe may then be added and the entire mixture incubated under hybridization conditions suitable to allow competitive hybridization to occur (e.g., 5 minutes at 65 C, then cooling to room temperature over a period of several hours in 2.3X SSC buffer). As those of skill in the art will recognize, the concentrations of the PCR product and fluorescent probe to be used may vary and may be 3o adjusted to optimize the reaction.

general, the concentrations of PCR product and fluorescent probe to be used are In adjusted so as to optimize the detectable loss of fluorescence resulting from competitive inhibition without sacrificing the ability of the assay to discriminate between perfect complementarity and one or more nucleotide mismatches. In an exemplary assay, the concentration of PCR product complementary to the oligonucleotide bound to the beads may be on the order of 1 to 10 times the molar concentration of fluorescent probe used. The fluorescent probe should preferably be added in an amount sufficient to achieve slightly less than saturation of the complementary oligonucleotide on the beads in order to obtain maximum sensitivity for competitive inhibition.
In a multiplexed assay employing the above principles, beadsets are separately prepared, pooled, and the bead-based hybridization analysis performed. In order to screen a given locus for mutations, beadset subsets are prepared such that subset 1 is coupled to a DNA segment identical to the wild type sequence, subset 2 is coupled to a DNA segment identical to a known is mutation 1 (which may represent a single or multiple point mutations, deletions or insertions), subset 3 is coupled to a DNA segment identical to a second known mutation 2, and so on. The subsets are then mixed to create a pooled headset.

When a nucleic acid sample is analyzed with such a beadset, only the bead subsets containing sequences identical to the test sample will show a large decrease in fluorescence (Fm).
Bead subsets containing unrelated or greatly disparate sequences will show little or no decrease in fluorescence (F,,,) and bead subsets containing very closely related sequences, such as point mutants, will show an intermediate decrease in fluorescence (Fm). Thus, a large decrease in the F. of only subset 1 would indicate homozygous wild-type; a large decrease in the F. of both subset 1 and subset 2 would indicate heterozygous wild-type / mutant 1 and so on. If the test sample is less inhibitory than the perfectly complementary sequence for any of the known sequences represented by the subsets then a new uncharacterized mutation is indicated. The test sample could then be sequenced to characterize the new mutation, and this sequence information used to construct a new subset for the beadset to detect the newly discovered mutation.

WO 97/14028 PCTIUS96t16198 The present invention has wide-spread advantages for detection of any of a number of nucleic acid sequences of interest in the genomic DNA of an individual or organism and has the advantages of being both rapid and extremely accurate in effecting the detection of such mutations.
The invention will find wide applicability in diagnosis of a number of genetically associated s disorders as well as in other applications where identification of genetic mutations may be important. Exemplary diseases include without limitation, diseases such as cystic fibrosis, generalized myotonia and myotonia congenita, hyperkalemic periodic paralysis, hereditary ovalocytosis, hereditary spherocytosis and glucose malabsorption; which are associated with mutations in the genes encoding ion transporters; multiple endocrine neoplasia, which is associated io with mutations in the MEN2a, b, and MEN1 genes; familial medullary thyroid carcinoma, and Hirschsprung's disease, which are associated with mutations in the ret proto-oncogene; familial hypercholesterolemia, which is associated with mutations in the LDL receptor gene;
neurofibromatosis and tuberous sclerosis, which are associated with mutations in the NFI gene, and NF type 2 gene; breast and ovarian cancer, which are associated with mutations in the BRCAI, 15 BRCA2, BRCA3 genes; familial adenomatous polyposis, which is associated with mutations in the APC gene; severe combined immunodeficiency, which is associated with mutations in the adenosine deaminase gene; xeroderma pigmentosum, which is associated with mutations in the XPAC gene; Cockayne's syndrome, which is associated with mutations in the ERCC6 excision repair gene; fragile X, which is associated with mutations in the finrl gene;
Duchenne's muscular 20 dystrophy, which is associated with mutations in the Duchenne's muscular dystrophy gene;
myotonic dystrophy, which is associated with mutations in the myotonic dystrophy protein kinase gene; bulbar muscular dystrophy, which is associated with mutations in the androgen receptor genes; Huntington's disease, which is associated with mutations in the Huntington's gene; Peutz-jegher's syndrome; Lesch-Nyhan syndrome, which is associated with mutations in the HPRT gene;
25 Tay-Sachs disease, which is associated with mutations in the HEXA gene;
congenital adrenal hyperplasia, which is associated with mutations in the steroid 21-hydroxylase gene; primary hypertension, which is associated with mutations in the angiotensin gene;
hereditary non-polyposis, which is associated with mutations in the hNMLH I gene; colorectal carcinoma, which is associated with mutations in the 2 mismatch repair genes; colorectal cancer, which is associated 30 with mutations in the APC gene; forms of Alzheimer's disease which have been associated with the apolipoprotein E gene, retinoblastoma, which is associated with mutations in the Rb gene; Li-Fraumeni syndrome, which is associated with mutations in the p53 gene; various malignancies and diseases that are associated with translocations: e.g., in the bcr/abl, bcl-2 gene; chromosomes I I to 14 and chromosomes 15 to 17 transpositions. The references at the end of the specification which may be referred to for further details describe genetic mutations associated with certain diseases which may be tested for in accordance with the invention as well as sequences provided in GENBANK.

Double Stranded Experiment to For the purposes of illustration, the two complementary strands of a double-stranded DNA
segment are referred to as strand "A" and strand "B". Either strand may be designated "A" or "B".
The wild-type "B" strand oligo (ras codon 12) having the oligonucleotide sequence 5'-GCCTACGCCACCAGCTCCAACTAC-3' (SEQ ID NO. 3) was coupled to 3.0 micrometers (gm) latex microspheres (manufactured by Interfacial Dynamics, Portland, OR) by carbodiimide coupling. Double stranded competitor was prepared by combining equal amounts of both the "A"
and "B" strands of either the wild-type or mutant version of the oligo, mutant "B" strand having the sequence 5'-GCCTACGCCACAAGCTCCAACTAC-3' (SEQ ID NO. 4) (ras codon 12) in 5X SSC buffer. Annealing was accomplished by heating the mixture to 65 C for five minutes.
then cooling slowly to room temperature. Competitive hybridization was accomplished by combining approximately 40 picomoles of the bead-attached oligo (wild-type "B"
strand) with the indicated amounts of double stranded competitor in 2.3X SSC buffer at approximately 25 C.
Finally, 100 picomoles of the fluorescinated oligo (wild-type "A" strand) was added to the reaction mixture. This mixture was incubated for two hours at room temperature, and then diluted with 300 pl of saline pH 7.3, and analyzed on the "FACSCAN' (manufactured by Becton-Dickinson Immunocytometry Systems, San Jose, CA). The results are shown in Table 20 below and in Figures 43a through 43c.

TABLE 20: Double-StrandedExperimental Results Using Wild-Type "B"
Oligonucleotide Double Stranded Percent Inhibition (%) Fold Competition Competitor (picomole) Wild-Type Mutant Wild-Type/Mutant 20 9 2.2 100 35 12 2.9 1000 56 17 3.3 These results clearly show that the DNA containing the single point mutation ("Mutant") was a detectably less effective inhibitor of hybridization between the DNA on the beads and the 5 fluorescent oligonucleotide probe at all concentrations of competitor tested.

Single Stranded Experiment The wild-type "B" strand oligo (ras codon 12) was coupled to 3.0 m latex microspheres (manufactured by Interfacial Dynamics) by carbodiimide coupling. Competitive hybridization was io accomplished by combining approximately 40 picomoles of the bead-attached oligo with 100 picomoles of the fluorescinated oligo (wild-type "A" strand) in 2.3X SSC
buffer. Finally, the indicated amounts of single stranded competitor (either mutant or wild-type) were added to two separate aliquots of the reaction mixture. These aliquots were incubated for two hours at room temperature, and then diluted with 300 l of saline pH 7.3. and analyzed on the FACSCAN flow t5 cytometer. The result of these experiments are set forth in Table 21 below and in Figures 44a and 44b.

TABLE 21: Single-Stranded Experimental Results Single Stranded Percent Inhibition (%) Fold Competition Competitor (picomole) Wild-Type Mutant Wild-Type/Mutant 100 "A" Strand 14 6 2.4 1000 "A" Strand 25 11 2.3 These results clearly show that the DNA containing the single point mutation ("Mutant") was a detectably less effective inhibitor of hybridization between the DNA on the beads and the s florescent oligonucleotide probe at all concentrations of competitor tested.

Resequencing analysis of PCR products using multiplexed analysis.
This example demonstrates the ability of flow cytometry to perform resequencing analysis of PCR products. As a model system, PCR products were derived from the DQAI
io gene, in the region of the gene which determines the major alleles of DQAI.
The DQA1 gene represents the DNA coding sequence for the alpha chain of the DQ molecule. DQ
is classified as a class II histocompatibility locus and is expressed in allelic form in all humans. Most individuals are heterozygous for DQA , i.e., they express two different DQA
alleles. The determination of DQA alleles is used in identity testing for paternity and forensic purposes.
is Seventeen alleles of DQAI have been defined by DNA sequencing; however, eight major alleles account for the large majority of the population. These alleles are determined by fourteen unique DNA sequences contained within four regions of the DQA1 gene;
all four regions are contained within a 227 base pair PCR product derived from human genomic DNA.

Flow cytometry was used to determine the presence or absence of all fourteen DNA
sequences in a PCR product simultaneously in a single reaction tube, thereby allowing determination of the DQA alleles expressed in a given sample. The system is based on competitive hybridization between the PCR product and complementary oligonucleotide pairs representing each of the fourteen unique DNA sequences. One strand of each oligonucleotide pair is coupled to a unique subset of microspheres and the complementary strand is labeled with a green emitting fluorophore. After coupling, the fourteen unique microsphere subsets were pooled to produce the mixed bead set. After addition of the fourteen fluorescent oligonucleotides and the PCR product to the beadset, the mixture is hybridized and then s analyzed by flow cytometry. The ability of the PCR product to inhibit the hybridization of the complementary fluorescent oligonucleotides to their respective microsphere subsets is used to determine the DNA sequences, and thus, the allele(s) present in the PCR
product.

Microspheres: Carboxylate-modified latex (CML) microspheres of 5.5 micron mean diameter io were obtained from Bangs Laboratories, Inc. (Carmel, IN). The microspheres were differentially dyed with varying concentrations of two fluorescent dyes with orange and red emission spectra to produce fourteen unique microsphere subsets.

Oligonucleotides: Fourteen oligonucleotide pairs (complementary strands designated "A" and is "B") corresponding to allelic sequences within the DQA1 gene (Table 22) were synthesized by Oligos, Etc. (Wilsonville, OR). using standard automated techniques. Each eighteen-base oligonucleotide was substituted at the 5' end with an amino-terminal linker during synthesis.
Oligonucleotide coupling to microspheres: The "B" strand of each oligonucleotide pair was 20 coupled to a unique subset of CML microspheres using carbodiimide chemistry. Briefly, 0.1 mL of a 1 mM solution of oligonucleotide in 0.1 M MES (2-[N-morpholino]ethanesulfonic acid), pH 4.5 was added to 1.0 mL of microspheres (1% solids) in 0.1 M MES, pH
4.5. To this mixture, 0.05 mL of a 10 mg/mL solution of EDC (1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride) was added and mixed vigorously. The mixture was incubated for 25 30 minutes at room temperature, followed by another addition of EDC, mixing, and incubation as above. Following the second incubation period, the microspheres were pelleted by centrifugation and resuspended in 0.4 mL of 0.1 M MES, pH 4.5 and stored at 4 C.
-Qligonucleotide labeling: The "A" strand of each oligonucleotide pair was fluorescently labeled 30 with Bodipy FL-X (6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) amino)hexanoic acid, succinimidvl ester) (Molecular Probes, Inc. , Eugene, OR). Briefly, a 400 tL solution containing 20 p.M oligonucleotide in 0.1 M sodium bicarbonate and 5% DMSO, pH
8.2 was reacted with 30 L Bodipy FL-X (10 mg/mL in DMSO) for 16-18 hours at room temperature. The mixture was desalted on a PD10 column equilibrated in TE (10 mM TrisHCl.

1 mM ethylenediamine tetraacetic acid (EDTA), pH 8.0) to remove excess unreacted dye and stored at 4 C.

TM
DNA extraction: Tissue sample (template) DNA was purified using the QIAmp Blood Kit (Qiagen, Chatsworth, CA) for DNA purification. Briefly, I x 107 tissue culture cells or 200 L
to whole blood is lysed with Qiagen protease and Buffer AL. The lysate is incubated at 70 C for minutes followed by addition of 210 L ethanol. The mixture is applied to a QIAmp spin column and centrifuged at 8,000 x g for 1 minute. The filtrate is discarded, 500 L Buffer AW
is added to the column and the centrifugation is repeated; this step is repeated. The filtrates are discarded and the DNA is eluted into a new tube by addition of 200 L Buffer AE, incubation at is room temperature for 1 minute, followed by centrifugation as above.

Polymerase chain reaction (PCR: PCR primers designated DQA AMP-A (5'-ATGGTGTAAA
CTTGTACCAGT-3', SEQ ID NO. 5) and DQA AMP-B (5'-TTGGTAGCAG
CGGTAGAGTTG-3', SEQ ID NO. 6) (World Health Organization, 1994) were synthesized by Oligos, Etc. (Wilsonville, OR) using standard automated techniques. PCR was performed with reagents (PCR buffer, dN T Ps, MgC12, and TAQ DNA polymerase) from Life Technologies, Inc.(Gaithersburg, MD). The reaction mixture (50 L) contained I M of each primer, 200 nM
dNTPs, 3 mM MgCl-,, 4 - 10 g/ml- DNA template, and 2.5 units TAQ DNA
polymerase in PCR buffer. The PCR reaction was performed on an Idaho Technologies thermal cycler (Idaho Falls, ID) using and initial step at 94 C for 45 sec, and 32 cycles of 94 C
for 30 sec, 48 C for 60 sec, and 72 C for 60 sec followed by a final hold at 72 C for 7 minutes.
Production of the product was verified by agarose electrophoresis and was quantified by size exclusion TM
chromatography on a Superdex 75 (10/30) column (Pharmacia, Piscataway, NJ).
The PCR
product was used without purification.

Competitive hybridization analysis: The hybridization reaction was performed in a total volume of 40 gL, containing approximately 8,000 of each bead subset for a total of approximately 110,000 microspheres, 50 nM of each fluorescent oligonucleotide, and 10 - 200 nM PCR
product, as competitor, in hybridization buffer (3 M trimethyl ammonium chloride, 0.15%
sodium dodecyl sulfate, 3 mM EDTA, and 75 mM TrisHCl, pH 8.0). Briefly, the beadset mixture, in hybridization buffer, was equilibrated at 55 C. The mixture of fluorescent oligonucleotides and PCR product was denatured in a boiling water bath for 10 minutes followed by quick-chilling on ice for 2 minutes. The microspheres were added, mixed well, and the entire reaction was allowed to hybridize for 30 minutes at 55 C. Following hybridization, io the mixture was diluted to 250 gL using hybridization buffer and analyzed by flow cytometry.
Results Microspheres for multiple analytes: Figure 45 illustrates the classification, using orange and red fluorescence, of the fourteen microsphere subsets used in the DQA1 analysis.
Each distinct microsphere subset bears one of the fourteen unique oligonucleotide capture probes on its surface. The level of green fluorescence associated with each subset, after hybridization with the fluorescent oligonucleotide probes, is also determined simultaneously, and measures the reactivity of the fluorescent oligonucleotides (and therefore, the reactivity of the PCR product) with each unique oligonucleotide sequence.
Titration of fluorescent oligonucleotide: To optimize the system for detection of PCR products, fluorescent oligonucleotide was titered in the presence or absence of PCR
competitor. Figure 46 illustrates the hybridization of increasing concentrations of fluorescent oligonucleotide "5503A" to microspheres coupled to oligonucleotide "5503B" in the presence or absence of a 200 nM concentration of double-stranded 0301 PCR product which contains the 5503 sequence.
In the absence of competitor, the level of "5503A" which hybridizes to the microspheres, detected as FL1, increases in a linear manner and reaches saturation at approximately 10 nM. In the presence of competitor, the binding curve is shifted to the right indicating inhibition of "5503A" hybridization.

_100-Concentration dependence of inhibition and detection of point mutations:
Figure 47 illustrates the inhibition of fluorescent oligonucleotide hybridization by varying concentrations of complementary and point mutant competitors in the presence of a fixed concentration of fluorescent oligonucleotide. The solid lines show the inhibition of hybridization to bead "3401 B" induced by competitors 3401 (U) or 3402 (n). The dashed lines show inhibition of hybridization to bead "3402B" induced by competitors 3401(s) or 3402 (I). Even at the lowest competitor concentration (10 nM), there is approximately a two-fold difference between the reactivity of the identical sequence versus the point mutant.

io Specificity of the multiple anal a assay: The specificity of the reaction of each DNA competitor sequence with the multiplexed microsphere subsets is illustrated in Table 23 and Figure 48, using double-stranded oligonucleotide competitors. The pattern of reactivity is consistent with the homology of the different oligonucleotides with identical sequences showing maximal reactivity, closely related sequences showing less reactivity, and unrelated sequences showing little or no reactivity.

Allele-specific reactivity patterns: In order to establish the reactivity patterns of the DQA1 alleles in a model system, simulated alleles were prepared by mixing the oligonucleotides representing the DNA sequences that would be present within a single PCR
product for a given allele. Figure 49 illustrates the typing of four simulated alleles of DQAI. By comparison to the allele reactivity chart shown in Table 24, it can be seen that each of the simulated alleles types correctly.

Typing of homozygous genomic DNA: To verify the ability of flow cytometry to correctly type PCR products prepared from genomic DNA, samples of DNA of known, homozygous type were obtained from the UCLA Tissue Typing Laboratory, Los Angeles, CA.
After PCR
amplification, these samples were typed using flow cytometry; the results are shown in Figure 50. By comparison to the allele reactivity chart (Table 24), it can be seen that the system correctly types these samples.

Typing of heterozygous genomic DNA: To determine the ability of multiplexed flow analysis to accurately type heterozygous DQA1 haplotypes, twenty-five samples of known heterozygous DQA1 type were obtained from the UCLA Tissue Typing Laboratory, Los Angeles, CA. The samples of homozygous DNA used above were added to the panel and all of the samples were coded and typed in a blinded study. The data from this study are presented in Table 25. The last column of Table 25 entitled "Type" indicates whether the haplotype indicated by UCLA
and the Luminex analysis agreed. In 34 of 35 samples, the haplotypes reported by both laboratories agreed; sample number 19 was not typed by the UCLA laboratory, but typed clearly as an 0501/0201 heterozygote in the Luminex analysis. Thus, the multiplexed analysis is io capable of typing the DQA1 haplotypes with at least 97% accuracy.

These studies have demonstrated that flow cytometry can rapidly and accurately perform resequencing ahalysis of PCR products. The model system used here required the analysis of fourteen DNA sequences to determine eight different DQA 1 alleles. Flow cytometry was able i5 to perform this analysis in a true simultaneous format, using a single sample of a single PCR
product in a single reaction tube. The entire analysis, including setup, hybridization, flow analysis, and data collection and analysis can be accomplished within an hour after PCR
amplification of the DNA sample. Thus, it is possible to perform tissue typing or other genetic analysis in less than three hours after obtaining a sample of blood, tissue, etcetera, including the 20 time required for extraction of DNA and PCR amplification.

TABLE 22: DQA1 DNA Sequences Name Sequence "A" Strand Sequence "B" Strand Allele Specificities DQA2501 TGGCCAGTACACCCATGA TCATGGGTGTACTGGCCA 0101, 0401, (SEQ ID NO. 7) (SEQ ID NO. 8) 0501 DQA2502 TGGCCAGTTCACCCATGA TCATGGGTGTACTGGCCA 0103, 0201, (SEQ ID NO. 9) (SEQ ID NO. 10) 0601 (SEQ ID NO. 11) (SEQ ID NO. 12) DQA3401 GAGATGAGGAGTTCTACG CGTAGAACTCCTCATCTC 0101, 0104 (SEQ ID NO. 13) (SEQ ID NO. 14) DQA3402 GAGATGAGCAGTTCTACG CGTAGAACTGCTCATCTC 0102, 0103, (SEQ ID NO. 15) (SEQ ID NO. 16) 0501 DQA3403 GAGACGAGCAGTTCTACG CGTAGAACTGCTCGTCTC 0401, 0601 (SEQ ID NO. 17) (SEQ ID NO. 18) DQA3404 GAGACGAGGAGTTCTATG CATAGAACTCCTCGTCTC 0201, 0301 (SEQ ID NO. 19) (SEQ ID NO. 20) DQA4101N ACCTGGAGAGGAAGGAGA TCTCCTTCCTCTCCAGGT 0101, 0102, (SEQ ID NO. 21) (SEQ ID NO. 22) 0201, 0301 (SEQ ID NO. 23) (SEQ ID NO. 24) DQA4103 ACCTGGGGAGGAAGGAGA TCTCCTTCCTCCCCAGGT 0401, 0501, (SEQ ID NO. 25) (SEQ ID NO. 26) 0601 DQA5501N TCAGCAAATTTGGAGGTT AACCTCCAAATTTGCTGA 0101, 0102, (SEQ ID NO. 27) (SEQ ID NO. 28) 0103 (SEQ ID NO. 29) (SEQ ID NO. 30) (SEQ ID NO.31) (SEQ ID NO. 32) DQA5504 TCAGACAATTTAGATTTG CAAATCTAAATTGTCTGA 0401, 0501, (SEQ ID NO. 33) (SEQ ID NO. 34) 0601 Table 23: Specificity of Oligonucleotide Inhibition Bead #

Oligo none 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
2501 64% 10% 5% -1% -6% 4% 1% 4% 1% 3% 5% -9% 2% 3%
2502 19% 77% -7% -2% 0% 1% -3% -3% -1% -4% -4% -4% -5% 4%
2503 7% 1% 85% 1% 2% 4% -3% -2% 4% -1% 1% -1% 5% 6%
3401 -1% -1% -3% 76% 1% 6% 1% 3% 2% 2% 5% -4% 2% 5%
3402 -1% -8% -12% 14% 83% 11% -5% -5% -3% -10% 0% -7% -4% 1%
3403 -2% -3% -1% 0% 7% 73% -1% 2% 1% -1% 5% -9% -4% 2%
3404 5% 5% 4% 6% 2% 8% 62% 10% 9% 6% 10% 0% 7% 9%
4101 6% 4% 6% 10% 5% 10% 6% 79% 18% 22% 31% 5% 12% 12%
4102 0% -1% -4% 0% -3% 5% 0% 8% 79% 3% 7% -1% 5% 4%
4103 -2% 11% 3% 5% 6% 7% 5% 4% 7% 71% 0% 8% 9% 6%
5501 -3% 3% 0% 2% 5% 5% 1% -1% 6% 1% 79% 9% 6% 4%
5502 3% 5% 5% 5% 7% 3% -1% 1% 2% -4% -7% 86% 4% 1%
5503 -5% 0% -6% 1%1 2% 0% -2% -8% 0% -7% -9% 13% 80% -5%
5504 3% 8% 9% 5% 6% --3-0/,F2%1 2% 7% 6% 4% 13% 4% 93%
TABLE 24. Allele Reactivity Chart Allele Pattern Sequence 0101 (1,1,1,1) 2501 3401 4101 5501 0102 (1,2,1,1) 2501 3402 4101 5501 0103 (2,2,2,1) 2502 3402 4102 5501 0201 (2,4,1,2) 2502 3404 4101 5502 0301 (3,4,1,3) 2503 3404 4101 5503 0401 (1,3,3,4) 2501 3403 4103 5504 0501 (1,2,3,4) 2501 3402 4103 5504 0601 (2,3,3,4) 2502 3403 4103 5504 Table 25. Blinded typing of Genomic DNA Samples for DQA1 BEAD SUBSET

1 4% 25% 35% 7% 69% 8% 58% 36% 61% -1% 78% 2% 75% -8% Y
2 13% -5% -7% 63% -4% -2% -3% 37% 2% -9% 77% 0% 1% -11% Y
3 19% -2% 40% 66% -3% 0% 65% 51% -2% -2% 78% -2% 76% -14% Y
4 22% I3% 18% 33% 68% 9% 1% -3% 6% 33% -14% 0%J-11%j 36% Y
38% 1% -7% 68% 78% 6% -1% 40% -1% 36% 76% -3% -5% 48% Y
6 -13% -10% 11% -10% -10% -7% 45% 21% -4% -12% -24% -9% 75% -19% Y
7 -7% 8% -6% -11% -8% 19% 27% 7% -5% 16% -27% 31%1 -6% 3M.[ Y
8 20% -5% -9% 1% 65% 53% -2% 37% -2% 30% 76% -2% -7% 43% Y
9 32% 5% -1% 7% 76% 5% 2% 57% 0% -1% 84% -3%1-11% -25% Y
32% 10% 60% 4% 72% 1% 71% 60% -4% 7% 83% -32% 76% -40% Y
% -6% -18% 44% Y
11 27% 7% 5% 10% 70% 5% 2% -14% -5% 45% -21 12 29% 3% 570/1 8% 71% 6% 67% 60% 0% 3% 84% -4% 77% -29% Y
13 25% 8% -2% 66% -4% -8% -8% 47% -1% -4% 83% -12%1-22%1-36%1 Y
14 16% 8% 33% -3% 0% 16% 29% 24% 2% 26% -25% -7% 47% 17% Y
7% 19% 8% -6% 26% 0% 31% 26% -5% 28% -31% 36%1-21% 230/61 Y
16 32% 9% 2% 18% 76% 8% 5% -1% -2% 48% -4% -6% -17% 52% Y
17 8-/.j 0%1 35% 6% 530/.j -4% 58% 45% 1% 48% -13% -1% 77% 41% Y
18 1% -2% 55% 3% 1% 1% 75% 54% 4% 6% -14% -3% 84% -12% Y
19 12% 18% 2% 5% 47% 8% 54% 37% 5% 46% -11% 46% 1% 39% ?
63% 15% 6% 42% 87% 17% 20% 64% 10% 65% 87% 6% 5% 60% Y
Y
21 440/61 62%1 8% 23% 76% 13% 73% 700/-l 1 20% 88% 64% 4% 10 2211 -6% 26% 5% 4% -3% 2% 55% 48% 6% -1% -7% 58% 1% -11% Y
23 56% 67% 13% 38% 87% 22% 24% 12% 75% 63% 89% 15% 15% 63% Y
24 15% 25% 5% 60% 58% 7% 10% 42% 61% -4% 90% -2% -3% -17% Y
47% 15% 12% 75% 15% 50% 17% 56% 4% 55% 83% 2% -5% 51% Y
26 30% 8% 56%1_15% 71% 5% 67% 65% 2% 9% 85% -1% 82%1-18%1 Y
27 13% 10% 10% 7% 27% 21% 5% 1% 3% 47% -16% 5% -5% 51% Y
28 23% 2% 0% 17% 23% 60% 14% -2% -1% 58% -18% -2% -3% 590/oY
Y
29 24% 6% 10% 48% 46% 5% 10% 56% 5% 8% 86% 2% -2%1-16%1--38% 12% 11% 73% 14% 48% 7% 55% 6% 55% 84% 1% -7% 50% Y
31 -1 % -1% 19% 0% 20% -1% 26% 29% -30/61 31% % -28% -4% 70% 31%1 Y
32 57% 16% 6% 83% 81% 11% 6% 59% 7% 60% 86% 9% -1% 53% Y
33 -13% 17% 24% 2% -1% 29% 47% 37% -6% 50% -11% 0% 80% 52% Y
34 33% 7% 5% 19% 75% r6% 13% 54% 1% 2% 85% 24% -3% -3% Y
-110/.] 19% 31% 10% -14% 0% 70% 46% -4% -2% -57% 44% 79% -8% Y -Measuring Enzymes with Bead-Based Assays The invention may also be used in several formats for measurement of enzymes, enzyme inhibitors and other analytes. For example, bead subsets can be generated that are modified with selected fluorescent substrates which can be enzymatically cleaved from the bead, resulting in a s loss of fluorescence (F,,,). Enzymes that can be detected and measured using the invention include but are not restricted to, protease, glycosidase, nucleotidase, and oxidoreductase. Any enzyme that results in selected bond cleavage can be measured. A cartoon of the action of enzyme on a bead-bound enzyme is shown in Figure 51a. An enzyme that acts upon a bead-bound substrate so that the bead-bound substrate becomes fluorescent or loses fluorescence i0 comprises an assay for the level of enzyme affecting such a change. Figures 51b and 51c depict these situations. Alteration of the substrate could be an oxidation or reduction, alteration of a chemical bond such a hydrolysis or other alteration of the bead-bound substrate so that the fluorescence of the substrate is altered in intensity or spectrum.

is Enzymes that act upon pro-enzymes (convertases) can be measured using a bead-bound substrate providing the reaction mixture contains the pro-enzyme and beads bearing a substrate that can be acted upon by the active form of the enzyme. (Providing the specificity of each activated enzyme is distinct, a multiplexed assay is achievable in which several pro-enzymes can be measured at the same time.) The sample is introduced into a mixture of pro-enzymes under 20 reaction conditions. After a fixed time interval during which the convertase acts upon the pro-enzyme, the beadsets specific for each enzyme generated from each pro-enzyme are added and the newly generated activities measured in a subsequent time period which is terminated when the beadsets are analyzed by flow cytometry. Such a process for a single pro-enzyme to enzyme conversion is illustrated by the cartoon in Figure 51d.
The action of the enzyme can be measured in an indirect but facile manner using a bead bound substrate as depicted in Figure 51e. The action of the enzyme on the bead-bound substrate results in the formation or revelation of a ligate for a fluorescent ligand present in the reaction mixture. The bead bearing the modified substrate then becomes fluorescent by virtue of 3o binding of the fluorescent ligand to the newly formed ligate. In practice, the enzyme(s) would be added to the beadset under reactive conditions. After a defined time period during which the enzyme acts upon the bead bound substrate, the enzyme action would be stopped and the fluorescent ligands added and after a period for association of ligand with the beadsets, the mixture analyzed by flow cytometry. The fluorescent ligands could be of a single reactivity or multiple ligands employed, the critical specificity is that of the enzyme for the substrate.

The bead-bound substrate may be used to detect the activation of enzyme when the enzyme requires a cofactor for activity. Under this circumstance, the level of the cofactor becomes the limiting component of the reaction mixture and determination of the level of io cofactor can be measured. Such a configuration is illustrated in Figure 51f. The reaction mixture contains the bead-bound substrate as well as the apo-enzyme. After introduction of the analyte (enzyme cofactor), the reaction mixture is held under reactive conditions for a fixed period of time followed by analysis of the beads by flow cytometry, the level of cofactor limits the level of enzyme activity. Providing the enzymes present require different cofactors and have action on different substrate-bearing beadsets, several cofactors could be measured in a single assay mixture.

In short, bead-borne substrates can be used as reagent as are soluble substrates for enzymes. However, because each type of bead bearing a unique substrate can be distinguished, a mixture of bead subsets can be used to measure several enzyme activities simultaneously in the same reaction mixture.

Fluids that can be analyzed using these techniques include plasma, serum, tears, mucus, saliva, urine, pleural fluid, spinal fluid and gastric fluid, sweat, semen, vaginal secretions, fluid from ulcers and other surface eruptions, blisters, and abscesses, and extracts of tissues including biopsies of normal, malignant, and suspect tissues. An assay according to this aspect of the invention proceeds as follows:

1. Beads containing reactive surface groups (one of the following: amino, aldehyde, acid chloride, amidine, phenolic hydroxyl, phenyl amine, carboxyl) are obtained that can be discriminated on the basis of, for example, forward angle light scatter, C f, right angle light scatter, C2, and one of several wavelengths of fluorescence C3 ... Cõ which are designated as orange and red fluorescence, for example, and comprise a number of subsets.
2. Subsets thus obtained are derivatized with a peptide (substrate) having a terminal fluorescent green group, for example fluorescein (F,,,).
3. Unreacted surface groups and hydrophobic surface of the bead are blocked and the subsets are processed by a particle analyzer and sorter (FACSCAN) and a uniform population of particles are separated which have a low coefficient of variance for F,,,.
(e.g., 3%).
4. A fluid to be tested is diluted with an appropriate buffer and added to the beadset mixture to allow enzymes present in the sample to react with (cleave) their corresponding substrate on the surfaces of the beads.
5. After a defined period of time, the reaction is stopped and the entire mixture processed by a flow cytometer and results are determined.

is The presence of an enzyme in the clinical sample is indicated by loss of fluorescence resulting from the cleavage of the fluorescent Fm substrate from the bead surface. Because the beads are analyzed in a very small volume (e.g., about 6 picoliters) as they are passed through the flow cytometer's laser beam, interference from free fluorescent molecules (cleaved substrate) will not significantly interfere with the assay. This obviates the requirement of washing of the beads prior to assay and simplifies the procedure significantly.

Time Time measurement is an important feature of the analysis. The essence of the measurement of an enzyme activity is a change in substrate with time. The activity can be determined by setting a period of time during which the clinical sample is in contact with the beads using standard conditions of pH, ionic composition and temperature. Two alternative processes are available for determination of the bead-bound substrate with time, that is the time expired while the enzyme(s) is (are) acting on each beadset(s).

External Time In this configuration, as each bead is measured by the flow cytometer, the time at which each measurement was obtained is recorded along with the bead's other measurements. Prior to the beginning of the assay, the baseline measurement is determined. Once the enzyme (clinical sample) is added to the bead mixture, the sample analysis begins. As the beads proceed through the instrument, the time data collected is used to determine the length of time that the bead has been exposed to the clinical sample. The Fm data collected over the period of the assay is used to determine the rate of change of substrate on the beads (kinetics) and thus the rate readily derived for each bead subset in the mixture exposed to the clinical sample.
Internal Time Time can be determined and at the same time a quality control internally generated by including a "timer" bead subset that bears a substrate which is acted on by an enzyme that does not naturally occur in the clinical sample to be tested. The use of non-pathogenic microbial is enzymes and substrates with human samples, for example, would suffice. The corresponding "timer" enzyme is added to the dilution buffer so that a known concentration of the "timer"
enzyme is present in the buffer. The degree of action of the "timer" enzyme upon the beads in the "timer" subset can be measured as a function of the loss of fluorescence of the beads in the subset to ensure that proper reaction conditions are achieved. The level of fluorescence of the timer beads can thus be used as an internal standard and an estimation of time.

Determination of Enzyme Inhibitors or Regulators In addition to direct assay of enzymes, an assay of this type may also be used to detect enzyme inhibitors or regulators. In accordance with this variation, samples being tested for inhibitors are added to the beadset followed by the corresponding enzymes. If inhibitors are present, the measured fluorescent (Fm) values will not be decreased to the same extent as a control containing no inhibitors. In accordance with Figure 52, in a similar manner, inhibitors of enzyme activators or binders of cofactors can be measured.

The present invention provides numerous advantages and overcomes many problems associated with prior art techniques of multiplexed diagnostic and genetic analysis apparatus and methods. It will be appreciated by those of ordinary skill having the benefit of this disclosure that numerous variations from the foregoing illustration will be possible without departing from the inventive concept described herein. Accordingly, it is the claims set forth below, and not merely the foregoing illustration, which are intended to define the exclusive rights claimed in this application program.

REFERENCES
1. Hum. Biol. 64: 167-174 (1992) Mutation in Cystic Fibrosis: a Review Spatial Distribution of the DF508. DeBraekeleer, M. and Daigeneault, J.;
2. Science 257: 797-800 (1992) [92358240] The skeletal muscle chloride channel in dominant and recessive human myotonia. M. C. Koch, K. Steinmeyer, C. Lorenz, K.
Ricker, F. Wolf, M. Otto, B. Zoll, Lehmann-Horn, K. H. Grzeschik & T. J.
Jentsch;
3. Neuron 12: 281-94 (1994) [94153549] Sodium channel mutations in paramyotonia congenita uncouple inactivation from activation. M. Chahine, A. L. George, M.
Zhou, S.
Ji, W. Sun, R. L. Barchi & R. Horn. Ann. Neurol. 33: 300-7 (1993) [93270429];
Sodium channel mutations in paramyotonia congenita and hyperkalemic periodic paralysis. L. J.
Ptacek, L. Gouw, H. Kwiecinski, P. McManis, J. R. Mendell, R. J. Barohn, A. L.
George, R. L. Barchi, M. Robertson & M. F. Leppert;
4. Ann. Neurol. 33: 300-7 (1993) [93270429] Sodium channel mutations in paramyotonia congenita and hyperkalemic periodic paralysis. L. J. Ptacek, L. Gouw, H.
Kwiecinski, P.
McManis, J. R. Mendell, R. J. Barohn, A. L. George, R. L. Barchi, M. Robertson & M. F.
Leppert; Cell 67: 1021-7 (1991) [92069747] Identification of a mutation in the gene causing hyperkalemic periodic paralysis. L. J. Ptacek, A. L. George, R. C.
Griggs, R.
Tawil, R. G. Kallen, R. L. Barchi, M. Robertson & M. F. Leppert;
5. Nature 355: 836-8 (1992) [92168137] Defective anion transport activity of the abnormal band 3 in hereditary ovalocytic red blood cells. A. E. Schofield, D. M.
Reardon & M. J.
Tanner, _110-6. J. Clin. Invest. 93: 121-30 (1994) [94110314] Duplication of 10 nucleotides in the erythroid band 3 (AE 1) gene in a kindred with hereditary spherocytosis and band 3 protein deficiency (band 3PRAGUE). P. Jarolim, H. L. Rubin, S. C. Liu, M. R. Cho, V.
Brabec, L.
H. Derick, S. J. Yi, S. T. Saad, S. Alper, C. Brugnara et al.;
s 7. Acta Physiol. Scand. Suppl. 607: 201-7 (1992) [93080072] The Na+/glucose cotransporter (SGLT1). E. M. Wright, E. Turk, K. Hager, L. Lescale-Matys,B. Hirayama, S.
Supplisson & D. D. Loo. Nature 350: 354-6 (1991) [91179516]; Glucose/galactose malabsorption caused by a defect in the Na+/glucose cotransporter. E. Turk, B. Zabel, S.
Mundlos, J. Dyer & E. M. Wright;
8. Nature 363: 458-60 (1993) [93275414] Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A.L. M. Mulligan, J. B. Kwok, C. S. Healey, M. J.
Elsdon, C. Eng, E. Gardner, D.R. Love, S. E. Mole, J. K. Moore, L. Papi, et al.;
9. Nature 367: 375-6 (1994) [94159102] A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma [see is comments] R. M. Hofstra, R. M. Landsvater, I. Ceccherini, R. P. Stulp, T.
Stelwagen, Y.
Luo, B. Pasini, J. W. Hoppener, H. K. van Amstel, G. Romeo, et al.;
10. Nature 367: 378-80 (1994) [94159104] Mutations of the RET proto-oncogene in Hirschsprung's disease [see comments] P. Edery, S. Lyonnet, L. M. Mulligan, A.
Pelet, E.
Dow, L. Abel, S. Holder, C. Nihoul-Fekete, B. A. Ponder & A. Munnich; Nature 367: 377-8 (1994) [94159103] Point mutations affecting the tyrosine kinase domain of the RET
proto-oncogene in Hirschsprung's disease [see comments] G. Romeo, P.
Ronchetto, Y.
Luo, V. Barone, M. Seri, I. Ceccherini, B. Pasini, R. Bocciardi, M. Lerone, H.
Kaariainen, et al.;
11. Hum. Mutat. 1: 445-66 (1992) [93250847] Molecular genetics of the LDL
receptor gene in familial hypercholesterolemia. H. H. Hobbs, M. S. Brown & J. L. Goldstein;
Clin. Chem.
36: 900-3 (1990) [90291682] Use of polymerase chain reaction to detect heterozygous familial hypercholesterolemia. M. Keinanen, J. P. Ojala, E. Helve, K. Aalto-Setala, K.
Kontula & P. T. Kovanen;
12. Hum. Genet. 93: 351-2 (1994) [94171244] Two CA/GT repeat polymorphisms in intron 27 of the human neurofibromatosis (NF1) gene. C. Lazaro, A. Gaona & X. Estivill;
Am J

Hum Genet 54: 424-36 (1994) [94160989] Deletions spanning the neurofibromatosis 1 gene: identification and phenotype L. M. Kayes, W. Burke, V. M. Riccardi, R.
Bennett, P.
Ehrlich, A. Rubenstein & K. Stephens; Cell 75: 1305-15 (1993) [94094325]
Identification = and characterization of the tuberous sclerosis gene on chromosome 16. The European Chromosome 16 Tuberous Sclerosis Consortium;
13. Hum. Mol. Genet. 2: 1823-8 (1993) [94108432] Genetic analysis of the BRCA
I region in a large breast/ovarian family: refinement of the minimal region containing BRCA1. D. P.
Kelsell, D. M. Black, D. T. Bishop & N. K. Spurr;
14. Hum. Mutat. 3: 12-8 (1994) [94163183] Exon eight APC mutations account for a to disproportionate number of familial adenomatous polyposis families. D. J.
Koorey, G. W.
McCaughan, R. J. Trent & N. D. Gallagher, Hum. Mutat. 1: 467-73 (1992) [93250848]
Screening for germ-line mutations in familial adenomatous polyposis patients:
61 new patients and a summary of 150 unrelated patients. H. Nagase, Y. Miyoshi, A.
Horii, T.
Aoki, G. M. Petersen, B. Vogelstein, E. Maher, M. Ogawa, M. Maruyama, J.
Utsunomiya, et al.; Cell 66: 589-600 (1991) [91330306] Identification and characterization of the familial adenomatous polyposis coli gene. J. Groden, A. Thliveris, W.
Samowitz, M.
Carlson, L. Gelbert, H. Albertsen, G. Joslyn, J. Stevens, L. Spirio, M.
Robertson, et al.;
15. Hum. Mol. Genet. 2: 1307-8 (1993) [94004878] A missense mutation in exon 4 of the human adenosine deaminase gene causes severe combined immunodeficiency. U.
Atasoy, C. J. Norby-Slycord & M. L. Markert; Hum. Mol. Genet. 2: 1099-104 (1993) [94004847]
The interleukin-2 receptor gamma chain maps to Xg13.1 and is mutated in X-linked severe combined immunodeficiency, SCIDXI J. M. Puck, S. M. Deschenes, J. C. Porter, A. S.
Dutra, C. J. Brown, H. F. Willard & P. S. Henthorn; Cell 73: 147-57 (1993) [93214986]
Interleukin-2 receptor gamma chain mutation results in X-linked severe combined immunodeficiency in humans. M. Noguchi, H. Yi, H. M. Rosenblatt, A. H.
Filipovich, S.
Adelstein, W. S. Modi, O. W. McBride & W. J. Leonard, Am. J. Med. Genet. 42:

(1992) [92125333] Five missense mutations at the adenosine deaminase locus (ADA) detected by altered restriction fragments and their frequency in ADA--patients with severe combined immunodeficiency(ADA-SCID). R. Hirschhorn, A. Ellenbogen & S. Tzall;

16. Mutat. Res. 273: 193-202 (1992) [92186915] Three nonsense mutations responsible for group A xeroderma pigmentosum. I. Satokata, K. Tanaka, N. Miura, M. Narita, T.
Mimaki, Y. Satoh, S. Kondo & Y. Okada; J. Biol. Chem. 266: 19786-9 (1991) [92011785]
Identification and characterization of xpac protein, the gene product of the human XPAC
(xeroderma pigmentosum group A complementing) gene. N. Miura, I. Miyamoto, H.
Asahina, I. Satokata, K. Tanaka & Y. Okada;
17. Nucleic Acids Res. 21: 419-26 (1993) [93181229] Structure and expression of the excision repair gene ERCC6, involved in the human disorder Cockayne's syndrome group B.
C.
Troelstra, W. Hesen, D. Bootsma & J. H. Hoeijmakers;
to 18. Am. J. Hum. Genet. 51: 299-306 (1992) [92351926] A microdeletion of less than 250 kb, including the proximal part of the FMR-I gene and the fragile-X site, in a male with the clinical phenotype of fragile-X syndrome. D. Wohrle, D. Kotzot, M. C. Hirst, A. Manca, B. Korn, A. Schmidt, G. Barbi, H. D. Rott, A. Poustka, K. E. Davies, et al.;
19. Lancet 341: 273-5 (1993) [93148721] Direct diagnosis of carriers of point mutations in Duchenne muscular dystrophy. S. C. Yau, R. G. Roberts, M. Bobrow & C. G.
Mathew.
Hum. Genet. 90: 65-70 (1992) [93052247] Molecular genetic analysis of 67 patients with Duchenne/Becker muscular dystrophy. S. Niemann-Seyde, R. Slomski, F.
Rininsland, U.
Ellermeyer, J. Kwiatkowska & J. Reiss. Hum. Genet. 84: 228-32 (1990) [90152651 ] Rapid detection of deletions in the Duchenne muscular dystrophy gene by PCR
amplification of deletion-prone exon sequences M. Hentemann, J. Reiss, M. Wagner & D. N.
Cooper, Nature 322: 73-7 (1986) [86257412] Analysis of deletions in DNA from patients with Becker and Duchenne muscular dystrophy. L. M. Kunkel;
20. Genomics 18: 673-9 (1993) [94140369] Genomic organization and transcriptional units at the myotonic dystrophy locus. D. J. Shaw, M. McCurrach, S. A. Rundle, H. G.
Harley, S.
R. Crow, R. Sohn, J. P. Thirion, M. G. Hamshere, A. J. Buckler, P. S. Harper, et al. Arch.
Neurol. 50: 1173-9 (1993) [94029649] The myotonic dystrophy gene. A. Pizzuti, D. L.
Friedman & C. T. Caskey; Hum. Mol. Genet. 2: 299-304 (1993) [93271990]
Structure and genomic sequence of the myotonic dystrophy (DM kinase) gene. M. S. Mahadevan, C.
Amemiya, G. Jansen, L. Sabourin, S. Baird, C. E. Neville, N. Wormskamp, B.
Segers, M.
Batzer, J. Lamerdin, et al.;

21. Nature 352: 77-9 (1991) [91287825] Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. A. R. La Spada, E. M. Wilson, D. B. Lubahn, A. E.
Harding & K. H. Fischbeclc Neurology 42: 2300-2 (1992) [93096171] Strong correlation between the number of CAG repeats in androgen receptor genes and the clinical onset of features of spinal and bulbar muscular atrophy. S. Igarashi, Y. Tanno, O. Onodera, M.
Yamazaki, S.
Sato, A. Ishikawa, N. Miyatani, M. Nagashima, Y. Ishikawa, K. Sahashi, et al.;
Science 256: 784-9 (1992) [92271195] Triplet repeat mutations in human disease. C. T.
Caskey, A.
Pizzuti, Y. H. Fu, R. G. Fenwick & D. L. Nelson;
22. Hum. Mol. Genet 2: 1713-5 (1993) [94093563] Analysis of the huntingtin gene reveals a trinucleotide-length polymorphism in the region of the gene that contains two CCG-rich stretches and a correlation between decreased age of onset of Huntington's disease and CAG repeat number. D. C. Rubinsztein, D. E. Barton, B. C. Davison & M. A.
Ferguson-Smith; Mol. Cell. Probes 7: 235-9 (1993) [93375991] A new polymerase chain reaction (PCR) assay for the trinucleotide repeat that is unstable and expanded on Huntington's disease chromosomes. J. P. Warner, L. H. Barron & D. J. Brock; Cell 72: 971-83 (1993) [93208892] A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group;
23. J. Clin. Invest 83: 11-3 (1989) [89093407] Identification of a single nucleotide change in the hypoxanthine-guanine phosphoribosyltransferase gene (HPRTYale) responsible for Lesch-Nyhan syndrome. S. Fujimori, B. L. Davidson, W. N. Kelley & T. D.
Palella; Proc.
Natl. Acad. Sci. U.S.A. 86: 1919-23 (1989) [89184538] Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA. R. A. Gibbs, P. N. Nguyen, L. J. McBride, S. M. Koepf & C. T. Caskey;
Genomics 7: 235-44 (1990) [90269813] Multiplex DNA deletion detection and exon sequencing of the hypoxanthine phosphoribosyltransferasegene in Lesch-Nyhan families.
R. A. Gibbs, P. N. Nguyen, A. Edwards, A. B. Civitello & C. T. Caskey;
24. Nature 333: 85-6 (1988) [88202110] Identification of an altered splice site in Ashkenazi Tay-Sachs disease. E. Arpaia, A. Dumbrille-Ross, T. Maler, K. Neote, M.
Tropak, C.
Troxel, J. L. Stirling, J. S. Pitts, B. Bapat, A. M. Lamhonwah, et al.; J.
Biol. Chem. 263:

18587-9 (1988) [89066640] The major defect in Ashkenazi Jews with Tay-Sachs disease is an insertion in the gene for the alpha-chain of beta-hexosaminidase R.
Myerowitz & F. C.
Costigan; Hum. Mutat. 1: 303-9 (1992) [93250824] A mutation common in non-Jewish Tay-Sachs disease: frequency and RNA studies. B. R. Akerman, J. Zielenski, B.
L. Triggs-Raine, E. M. Prence, M. R. Natowicz, J. S. Lim-Steele, M. M. Kaback, E. H.
Mules, G. H.
Thomas, J. T. Clarke, et al.;
25. Clin. EndocrinoL (Oxf) 38: 421-5 (1993) [93306853] Prenatal diagnosis of congenital adrenal hyperplasia by direct detection of mutations in the steroid 21 -hydroxylase gene. G.
Rumsby, J. W. Honour & C. Rodeck; Proc. Natl. Acad. Sci. U.S.A. 90: 4552-6 (1993) [93281617] Mutations in the CYP 11 B 1 gene causing congenital adrenal hyperplasia and hypertension cluster in exons 6, 7, and 8. K. M. Curnow, L. Slutsker, J.
Vitek, T. Cole, P.
W. Speiser, M. I. New, P. C. White & L. Pascoe; Hum. Genet. 89: 109-10 (1992) [92250001 ] Prenatal diagnosis of 2 1 -hydroxylase deficiency congenital adrenal hyperplasia using the polymerase chain reaction. D. Owerbach, M. B. Draznin, R. J.
Carpenter & F.
Greenberg;
26. Nucleic Acids Res. 20: 1433 (1992) [92220641] PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene (DCP 1) (dipeptidyl carboxypeptidase 1). B. Rigat, C. Hubert, P. Corvol & F. Soubrier, Biochem.
Biophys.
Res. Commun. 184: 9-15 (1992) [92231988] Association of a polymorphism of th e angiotensin I-converting enzyme gene with essential hypertension. R. Y. Zee, Y. K. Lou, L. R. Griffiths & B. J. Morris;
27. Nature 368: 258-61 (1994) [94195398] Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. C.
E.
Bronner, S. M. Baker, P. T. Morrison, G. Warren, L. G. Smith, M. K. Lescoe, M.
Kane, C.
Earabino, J. Lipford, A. Lindblom, et al.; Oncogene 9: 991-4 (1994) [94151027]
DNA
alterations in cells from hereditary non-polyposis colorectal cancer patients.
C. Wu, Y.
Akiyama, K. Imai, S. Miyake, H. Nagasaki, M. Oto, S. Okabe, T. Iwama, K.
Mitamura, H.
Masumitsu, et al.;
28. Science 263: 1625-9 (1994) [94174309] Mutation of a mutL homolog in hereditary colon cancer [see comments] N. Papadopoulos, N. C. Nicolaides, Y. F. Wei, S. M.
Ruben, K. C.

Carter, C. A. Rosen, W. A. Haseltine, R. D. Fleischmann, C. M. Fraser, M. D.
Adams, et al.; Cell 75: 1215-25 (1993) [94084796] Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. F. S. Leach, N. C. Nicolaides, N.
Papadopoulos, B. Liu, J.
Jen, R. Parsons, P. Peltomaki, P. Sistonen, L. A. Aaltonen, M. Nystrom-Lahti ;
29. Hum. Mutat. 3: 12-8 (1994) [94163183] Exon eight APC mutations account for a disproportionate number of familial adenomatous polyposis families. D. J.
Koorey, G. W.
McCaughan, R. J. Trent & N. D. Gallagher, Hum. Mutat. 2: 478-84 (1993) [94154735]
Simple, rapid, and accurate determination of deletion mutations by automated DNA
sequencing of heteroduplex fragments of the adenomatous polyposis coli (APC) gene to generated by PCR amplification. K. Tamura, Y. Yamamoto, Y. Saeki, J.
Furuyama & J.
Utsunomiya;
30. Biochim. Biophys. Acta 1155: 43-61 (1993) [93277907] Molecular characterization of the retinoblastomasusceptibilitygene. D. W. Goodrich& W. H. Lee; Br. J. Cancer 68, (1993) Mechanisms of oncogenesis in patients with familial retinoblastoma Onadim,Z., Hogg,A. & J.K Cowell;
31. Cancer Res. 54: 1298-304 (1994) [94163623] Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li-Fraumeni families. J. M. Birch, A. L.
Hartley, K.
J. Tricker, J. Prosser, A. Condie, A. M. Kelsey, M. Harris, P. H. Jones, A.
Binchy, D.
Crowther, et al.;
32. Leukemia 8: 186-9 (1994) [94118546] An optimized multiplex polymerase chain reaction (PCR) for detection of BCR-ABL fusion mRNAs in haematological disorders. N. C.
Cross, J. V. Melo, L. Feng & J. M. Goldman; Blood 69: 971-3 (1987) [87129392] bcr-abl RNA
in patients with chronic myelogenous leukemia. E. Shtivelman, R. P. Gale, O.
Dreazen, A. Berrebi, R. Zaizov, I. Kubonishi, I. Miyoshi & E. Canaani bcl-2; Diagn.
Mol. Pathol.
2: 241-7 (1993) [94163382] Rearrangement of the BCL-2 gene in follicular lymphoma.
Detection by PCR in both fresh and fixed tissue samples. J. Liu, R. M. Johnson & S. T.
Traweek; Blood 83: 1079-85 (1994) [94154269] Cytometric detection of DNA
amplified = with fluorescent primers: applications to analysis of clonal bcl-2 and IgH
gene rearrangements in malignant lymphomas. R. L. Barker, C. A. Worth & S. C.
Peiper; Br. J.
Cancer 67: 922-5 (1993) [93264208] Detection of bcl-2/JH rearrangement in follicular and diffuse lymphoma: concordant results of peripheral blood and bone marrow analysis at diagnosis. R. Yuan, P. Dowling, E. Zucca, H. Diggelmann & F. Cavalli;
33. Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989). J. Sambrook, E. Fritch, & T. Maniatis;
s 34. Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467 (1977), DNA Sequencing with Chain Terminating Inhibitors, F. Sanger, S. Niklen & A.R. Coulsen.

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(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single s (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

s (2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid to (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO 97/14028 PCT/US96(16198 (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs is (IB) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single s (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

(2) INFORMATION FOR SEQ ID NO:3 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs is (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 1:

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single s (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

is ATCTTCTAAA TCTGCGGA 18 (2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

s (2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

MICROFICHE APPENDIX A

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DatabaseName = "C:\ACCESS\orbit. MDB"
Exclusive = 0 'False Height 270 Left = 8160 Options = 0 ReadOnly = 0 'False SUBSTITUTE SHEET (RULE 26) RecordSource = "assay" -143-Top = 0 Visible 0 'False Width = 1455 End Begin ComboBox CubeSel Height = 300 Left = 120 Style = 2 'Dropdown List Tabindex = 30 Top = 960 Width = 1815 End Begin Data Datal Caption = "Datal"
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Font3D = 0 'None Height = 495 Left = 7920 Picture = (none) Tabindex = 21 Top = 5520 Width = 1455 End Begin SSCommand ResClear Caption = "Clear"
Font3D = 0 'None Height = 495 Left = 7920 Picture = (none) Tabindex = 20 Top = 4920 Width = 1455 End Begin Grid Gridi FixedCols 0 Height = 2295 SUBSTITUTE SHEET (RULE 26) Left = 2640 -144-Rows = 64 ScrollBars = 2 'Vertical Tabindex = 19 Top = 4440 Width = 5175 End Begin GRAPH Graphs AsciiSymbol = "0"
GraphCaption = "Classification"
GraphTitle = "Classification"
GraphType 9 'Scatter GridStyle = 3 'Horizontal and Vertical Height = 2295 Left = 120 NumPoints = 20 NumSets = 16 Tabindex = 18 Top = 4440 Width = 2415 End Begin SSPanel Panel3D2 Alignment = 0 'Left Justify - TOP
BackColor = &HOOCOCOCO&
Bevellnner = 1 'Inset Caption = "Operator Insturctions"
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Font3D = 0 'None Height = 375 Left = 8040 Picture = (none) TabIndex = 17 Top = 120 Width = 855 End End Begin TextBox SIBox Height = 1815 Left = 120 MultiLine = -1 'True TabIndex = 14 Top 1680 Width = 3735 End Begin SSPanel Panel3D1 Alignment = 0 'Left Justify - TOP
BackColor = &HOOCOCOCO&
Caption = "Cytometer Status"
Font3D = 0 'None Height = 2535 Left = 7680 TabIndex = 12 Top = 960 Width = 1815 Begin PictureBox FCPic BackColor = &HOOCOCOCO&
BorderStyle = 0 'None Height = 495 Index = 1 Left = 1320 Picture = (Icon) ScaleHeight = 492 ScaleWidth = 372 TabIndex = 25 Top = 240 Visible = 0 'False Width = 375 End Begin PictureBox FCPic BackColor = &HOOCOCOCO&
BorderStyle = 0 'None Height = 495 Index = 0 Left = 1320 Picture = (Icon) ScaleHeight = 492 ScaleWidth = 372 TabIndex = 24 Top = 240 Visible = 0 'False Width = 375 End Begin Label Labell0 Alignment = 1 'Right Justify BackStyle = 0 'Transparent Caption = "Pressure"

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Height = 255 Left = 0 Tabindex = 28 Top = 1560 Width = 1215 End Begin Label Label8 Alignment = 1 'Right Justify BackStyle = 0 'Transparent Caption = "Sheath Fluid"
Height = 255 Left = 120 Tabindex 27 Top = 960 Width = 1095 End Begin Label Labe17 Alignment = 1 'Right Justify BackStyle = 0 'Transparent Caption = "Flow Control"
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Font3D = 0 'None Height = 2535 Left = 3960 Tabindex = 4 Top = 960 Width = 1935 Begin SSCommand TCHalt Caption = "Counts"
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Enabled = 0 'False Font3D = 0 'None Height = 495 SUBSTITUTE SHEET (RULE 26) Left = 240 -147-Picture = (none) Tabindex = 6 Top = 1080 Width = 1455 End Begin SSCommand TCInit Caption = "Initialize"
Enabled = 0 'False Font3D = 0 'None Height = 495 Left = 240 Picture (none) Tabindex = 5 Top = 480 Width = 1455 End End Begin SSFrame MacCtl Caption = "Machine Control"
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Font3D = 0 'None Height = 495 Left = 240 Picture = (none) Tabindex = 10 Top = 1680 Width = 1335 End Begin SSCommand MCAdjust Caption = "Manual Adjust"
Font3D = 0 'None Height = 495 Left = 240 Picture = (none) Tabindex = 9 Top = 1080 Width = 1335 End Begin SSCommand MCCalib Caption = "Calibrate"
Font3D = 0 'None Height = 495 Left = 240 Picture = (none) Tabindex = 8 Top = 480 Width = 1335 End End Begin ComboBox AssaySel Height = 300 = Left = 2040 Style = 2 'Dropdown List SUBSTITUTE SHEET (RULE 26) TabIndex = 2 -148-Top = 960 Width = 1815 End Begin Label Label6 Alignment = 2 'Center BackStyle = 0 'Transparent BorderStyle = 1 'Fixed single Caption = "Results"
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Height = 255 Left = 120 TabIndex = 1 Top = 720 Width = 1575 End Begin Label Label3 Alignment = 2 'Center BackStyle = 0 'Transparent Caption = "Orbit Diagnostic operating system"
FontBold = -1 'True SUBSTITUTE SHEET (RULE 26) WO 97/14028 PC [US96/16198 Fontltalic = 0 'False FontName = "MS Sans Serif"
FontSize = 18 FontStrikethru = 0 'False FontUnderline = 0 'False ForeColor = &H00808000&
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ClientHeight = 6612 ClientLeft = 1212 ClientTop = 1128 ClientWidth = 5508 Height = 7032 Left = 1164 LinkTopic = "Form2"
MaxButton = 0 'False MinButton = 0 'False ScaleHeight = 6612 ScaleWidth = 5508 Top = 756 Width 5604 Begin SSFrame Frame3D5 Caption = "DDM Select"
Font3D = 3 'Inset w/light shading ForeColor = &H00000000&
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Caption = "Optionl"
Height = 255 Index = 1 Left = 600 TabIndex = 65 Top = 360 Width = 255 End Begin OptionButton Options BackColor = &H0000FF00&
Caption = "Optionl"
Height = 255 Index = 0 Left = 120 TabIndex = 64 Top 360 Value = -1 'True Width = 255 End Begin Shape Shape3 BackColor = &H000000FF&
BackStyle = 1 'opaque Height = 495 SUBSTITUTE SHEET (RULE 26) Left = 960 Top = 240 Width = 495 End Begin Shape Shape2 BackColor = &H000080FF&
BackStyle = 1 'Opaque Height = 495 Left = 480 Top = 240 Width 495 End Begin Shape Shapel BackColor = &HOO00FFOO&
BackStyle = 1 'Opaque Height = 495 Left = 0 Top = 240 Width = 495 End End Begin SSPanel Panel3D3 Alignment = 0 'Left Justify - TOP
BackColor = &HOOCOCOCO&
Bevellnner = 1 'Inset BorderWidth = 4 Font3D = 0 'None Height = 1095 Left = 4200 Tabindex = 56 Top = 2880 Width = 1335 Begin PictureBox Picturel BackColor = &HOOFFFFSO&
BorderStyle 0 'None Height = 855 Left = 120 Picture = (Icon) ScaleHeight = 852 ScaleWidth = 1092 Tabindex = 57 Top = 120 Width = 1095 Begin Data facset Caption = "Datal"
Connect = ""
DatabaseName = "C:\ACCESS\orbit.MDB"
Exclusive = 0 'False Height = 270 Left = 0 Options = 0 ReadOnly = 0 'False RecordSource = "facs_settings"
Top = 600 Visible = 0 'False Width = 1140 End Begin Label Labelll BackStyle = 0 'Transparent Caption = "ORBIT"
ForeColor = &H00808000&

SUBSTiME SHEET (RULE 26) Height = 255- 152 -Left = 360 Tabindex = 58 Top = 480 Width = 735 End End End Begin SSPanel Panel3D2 Alignment = 0 'Left Justify - TOP
BackColor = &HOOCOCOCO&
Bevellnner = 1 'Inset BorderWidth = 4 Font3D = 0 'None Height = 2655 Left = 4200 Tabindex 54 Top = 3960 Width = 1335 Begin SSCommand Command3D5 Caption = "Done"
Font3D 0 'None Height = 495 Left = 120 Picture = (none) Tabindex = 62 Top = 1920 Width = 1095 End Begin SSCommand Command3D4 Caption = "Save"
Font3D = 0 'None Hei:_ht = 495 Left = 120 Picture = (none) Tabindex = 61 Top = 1320 Width = 1095 End Begin SSCommand Command3D3 Caption = "Reset"
Font3D = 0 'None Height = 495 Left = 120 Picture = (none) Tabindex = 60 Top = 720 Width = 1095 End Begin SSCommand Command3D2 Caption = "Set"
Font3D = 0 'None Height = 495 Left 120 Picture (none) Tabindex = 59 Top = 120 Width = 1095 End Begin SSCommand Command3Dl Caption = "exit"
SUBSTITUTE SHEET (RULE 26) Font3D = 0 'None = Height = 375 Left = 240 Picture = (none) Tabindex = 55 Top = 2880 Width = 735 End End Begin SSPanel Panel3D1 Alignment = 0 'Left Justify - TOP
BackColor = &HOOCOCOCO&
Bevellnner = 1 'Inset BorderWidth 4 Caption = "Status"
Font3D = 0 'None Height = 2895 Left 4200 Tabindex = 53 Top = 0 Width = 1335 Begin Label Stat Alignment = 2 'Center BackStyle = 0 'Transparent Caption = "Labell5"
Height 255 Index = 3 Left = 120 Tabindex = 74 Top = 2520 Width = 1095 End Begin Label Labell5 Alignment = 2 'Center BackStyle = 0 'Transparent Caption = "DDN"
Height = 255 Left = 120 Tabindex = 73 Top = 2280 Width = 1095 End Begin Label Stat Alignment = 2 'Center BackStyle = 0 'Transparent Caption = "LabellSt' Height = 255 Index 2 Left = 120 Tabindex = 72 Top = 1920 Width = 1095 End Begin Label Stat Alignment = 2 'Center BackStyle = 0 'Transparent Caption = "Labell5"
Height = 255 = Index = 1 Left = 120 Tabindex = 71 SUBSTITUTE SHEET (RULE 26) Top = 1320 -154-Width = 1095 End Begin Label Stat Alignment = 2 'Center BackStyle = 0 'Transparent Caption = "Labell5"
Height = 255 Index = 0 Left = 120 Tabindex = 70 Top = 720 Width = 1095 End Begin Label Labell4 Alignment = 2 'Center BackStyle = 0 'Transparent Caption = "Samp. Volts"
Height = 255 Left. = 120 Tabindex = 69 Top = 1680 Width = 1095 End Begin Label Label13 Alignment = 2 'Center BackStyle = 0 'Transparent Caption = "Laser Amps"
Height = 255 Left = 120 Tabindex = 68 Top = 1080 Width = 1095 End Begin Label Labell2 Alignment = 2 'Center BackStyle = 0 'Transparent Caption = "Laser Volts"
Height = 255 Left = 120 Tabindex = 67 Top = 480 Width = 1095 End End Begin SSFrame Frame3D4 Caption = "DDM Amp"
Font3D = 3 'Inset w/light shading ForeColor = &H00000000&
Height = 975 Left = 0 Tabindex = 46 Top = 5640 Width = 2775 Begin HScrollBar ddmscroll Height = 255 Index = 0 LargeChange = 10 Left = 720 Max = 999 Tabindex = 51 SUBSTITUTE SHEET (RULE 26) Top = 240 - 155-W idth = 1335 End Begin TextBox ddmtxt Height = 285 Index = 0 Left = 2160 TabIndex = 50 Text = "Texts"
Top = 240 Width = 495 End Begin HScrollBar ddmscroll Height = 255 Index = 1 LargeChange = 10 Left = 720 Max = 999 TabIndex = 48 Top = 600, Width = 1335 End Begin TextBox ddmtxt Height = 285 Index = 1 Left = 2160 TabIndex = 47 Text = "Textl"
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Font3D = 3 'Inset w/light shading ForeColor = &H00000000&
Height = 1455 Left = 0 Tabindex = 8 Top = 1440 Width = 4215 Begin SpinButton Spina BackColor = &H000000FF&
Delay = 100 ForeColor = &HOOCOCOCO&
Height 495 Index = 4 Left = 3360 LightColor = &H000000CO&
ShadeColor = &H00000080&
ShadowBackColor = &H000000FF&
ShadowForeColor = &H000000FF&
SpinBackColor = &H000000FF&
SpinForeColor = &H00404040&
TdThickness = 2 Top = 720 Width = 495 End Begin SpinButton Spina BackColor = &H00008OFF&
Delay = 100 ForeColor = &HOOCOCOCO&
Height = 495 Index = 3 Left = 2640 LightColor = &H00008OFF&
ShadeColor &H00404080&
ShadowBackColor = &H00008OFF&
ShadowForeColor = &H00008OFF&
SpinBackColor = &H00008OFF&
SpinForeColor = &H00404040&
TdThickness = 2 Top = 720 Width = 495 End Begin SpinButton Spina BackColor = &HO000FFOO&
Delay 100 ForeColor = &HOOCOCOCO&
Height = 495 Index = 2 Left = 1920 LightColor = &HOOOOCOO0&
ShadeColor = &H00008000&
ShadowBackColor = &HO000FFOO&
ShadowForeColor = &HO000FFOO&
SpinBackColor = &HO000FFOO&
SpinForeColor = &H00404040&
TdThickness = 2 Top = 720 Width = 495 SUBSTITUTE SHEET (RULE 26) End Begin SpinButton Spina BackColor = &HOOFFFFOO&
Delay = 100 Height = 495 Index = 1 Left = 1080 SpinBackColor = &HOOFFFFOO&
TdThickness = 1 Top = 720 Width 495 End Begin SpinButton Spina BackColor = &HOO8OFF8O&
ForeColor = &HOOFFFFOO&
Height. = 495 Index = 0 Left = 360 LightColor = &HOOFFFF80&
ShadeColor &HOOFFFFSO&
ShadowForeColor = &HOOBOFF8O&
SpinBackColor = &HOOSOFF8O&
SpinForeColor = &H80000008&
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Top = 360 Width = 495 End Begin TextBox amp Height = 285 Index = 1 Left = 1080 Tabindex = 10 Text = "Texts"
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Top = 360 Width = 495 End Begin Label Label3 Alignment = 2 'Center BackStyle = 0 'Transparent Caption = "SSC"
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Height = 15 Index = 3 Left = 0 Tabindex = 18 Top = 240 Width = 495 ..End End Begin TextBox txtNumber Height = 285 Index = 4 Left = 3360 Tabindex = 5 Text = "Text1"
Top = 360 Width = 495 End Begin TextBox txtNumber Height 285 Index = 3 Left = 2640 Tabindex = 4 Text = "Texts"-Top = 360 Width = 495 SUBSTITUTE SHEET (RULE 26) End Begin TextBox txtNumber Height = 285 Index = 2 i,ei c = 1920 Tabindex = 3 Text = "Text?"
Top = 360 Width = 495 End Begin TextBox txtNumber Height = 285 Index = 1 Left 1080 Tabindex = 2 Text = "Text?"
Top = 360 Width = 495 End Begin SpinButton Spini BackColor = &HO000FFOO&
Delay = 100 ForeColor = &HOOCOCOCO&
Height = 495 Index 2 Left = 1920 LightColor = &H0000C000&
ShadeColor = &H00008000&
ShadowBackColor = &HO000FFOO&
ShadowForeColor = &HO000FFOO&
SpinBackColor = &HO000FFOO&
SpinForeColor = &H00404040&
TdThickness = 2 Top = 720 Width = 495 End Begin SpinButton Spin?
BackColor = &HOOFFFFOO&
Delay = 100 Height = 495 Index = 1 Left = 1080 SpinBackColor = &HOOFFFFOO&
TdThickness = 1 Top = 720 Width = 495 End Begin SSFrame PMT
Caption "Photo Multiplier"
Font's = 3 'Inset w/light shading Heig':: = 1455 Index = 0 Left = 0 Tabindex = 0 Top = 0 Width = 4215 Begin SpinButton Spin?
BackColor = &H000000FF&
Delay = 100 ForeColor = &HOOCOCOCO&
Height = 495 SUBSTITUTE SHEET (RULE 26) Index = 4 -167-Left = 3360 LightColor &H000000CO&
ShadeColor = &H00000080&
ShadowBackColor = &H000000FF&
ShadowForeColor = &H000000FF&
SpinBackColor = &H000000FF&
SpinForeColor = &H00404040&
TdThickness = 2 Top = 720 Width = 495 End Begin SpinButton Spin1 BackColor = &H00008OFF&
Delay = 100 ForeColor = &HOOCOCOCO&
Height = 495 Index = 3 Left = 2640 LightColor = &H00008OFF&
ShadeColor = &H00404080&
ShadowBackColor = &H00008OFF&
ShadowForeColor = &HOOOOSOFF&
SpinBackColor = &H00008OFF&
SpinForeColor = &H00404040&
TdThickness = 2 Top = 720 Width = 495 End Begin TextBox tx'-'Number Height 285 Index = 0 Left = 360 Tabindex = 1 Text = "Texts"
Top = 360 Width = 495 End Begin SpinButton Spini BackColor = &HOO8OFF8O&
ForeColor = &HOOFFFFOO&
Height = 495 Index = 0 Left = 360 LightColor = &HOOFFFFBO&
ShadeColor = &HOOFFFF8O&
ShadowForeColor = &HOO8OFF8O&
SpinBackColor = &HOO8OFF80&
SpinForeColor = &H80000008&
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Height = 255 Left = 1080 Tabindex = 7 Top = 1200 SUBSTITUTE SHEET (RULE 26) Width - 495 - 168 -End Begin Label Labell Alignment = 2 'Center BackStyle = 0 'Transparent Caption = "FSC"
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ix~.,~.*~~~=~~'.'}'4?:QJ_ :LW ='4 :;;: : ^1 Text `# 3 i> `' SUBSTITUTE SHEET (RULE 26) option Explicit Function vbgenproc () End Function Sub AssaySel Click () Dim y As Integer Dim x As Integer 'find assay record selected data2.Refresh Do While Not (AssaySel.Text = data2.Records et("assay name")) data2.Recordset.MoveNext Loop 'get number of events required by this assay events = data2.Recordset("event count") 'clear the testdef table; it holds the name of each token (biomolocule ' assayed) and the base green and over under values For x = 0 To 1023 For y = 0 To 1 testdef(y, x) = 0 Next y Next x datatst.Refresh 'now load the new values for this assay Do While Not datatst.Recordset.EOF
If AssaySel.Text = datatst.Records et("assay") Then x = datatst. Records et. (2) 'token value tkname(x) = datatst.Recordset("token_name") For y = 0 To I
testdef(y, x) = datatst.Recordset(y + 4) 'load over under' green and ba en Next y End If datatst.Recordset.MoveNext Loop If x = 0 Then MsgBox "There are no Measurement Parameters defined for this it, 48, "Assay Select"
lasttst = x End Sub Sub CubeSel Click () Dim y As Integer Dim x As Integer data2.Refresh AssaySel.Clear SUBSTITUTE SHEET (RULE 26) If CubeSel.Text = data2.Record- C~;" ='. . name") Then AssaySel.Addltem ecordset("assay_name") data2.Recordset.MoveNext Loop y = ReadPanel(tbuf(0)) loadpbuf For x = 13 To 42 If pbuf(x) <> tbuf(x) Then y = SendPanel(x, pbuf(x)) If y > 30 Then MsgBox "Flow Cytometer is not responding", 48, "Set Flow C
r"
Next x datahld.Refresh x = 0 Do While Not datahld.Recordset.EOF
If CubeSel.Text = datahid .Records at ("cube name") Then For y = 0 To 7 hldtab(y, x) = datahld.Recordset(y + 3) Next y x = x + 1 End If datahld.Recordset.MoveNext Loop If x = 0 Then MsgBox "There are no classification Parameters defined for be", 48, "Cube Select"
lastnode = x End Sub Sub Form Load ( ) Dim x As Integer 'x = InitBrd() datal.Refresh Do While Not datal.Recordset.EOP
CubeSel.Addltem datal. Records et cube-name .datal.Recordset.MoveNext Loop state = 0 gridl.ColWidth(0) = 2400 gridi.ColWidth(l) = 2600 For x = 1 To 63 gridl.RowHeight(x) = 500 Next x gridl.Row = 0 gridl.Col = 0 gridl.Text = "Biomolecule Assayed"
gridl.Col = 1 gridl.Text = "Result of Assay"
End Sub Sub MCAdjust Click O , If form1 CubeSel.Text <> "" Then Form2.Show End Sub SUBSTITUTE SHEET (RULE 26) Sub MCCalib Click O
Oplnst.Text = "Load Calibration Beads into FACS"
End Sub Sub MCEnd_Click () End End Sub Sub OpInstOK Click () state = state + 1 Select Case state Case 0 Case 1 Oplnst.Text = "Calibration Complete, Select Cube and Assay"
Case Else Oplnst.Text =
TClnit.Enabled = True End Select End Sub Sub ResClear_Click O
Dim x For x = 1 To 12 gridl.Row = x gridl.Rol = 0 gridl.Text = I'll gridl.Col = 1 gridl.Text =
Next x gridl.Row = 0 TCStart.Enabled = False End Sub Sub TCHalt_Click () during development, the halt button displays the results of classificati by token, **** token 0 is the reject class Dim x As Integer Dim y As Integer Dim z As Integer Dim measure As Integer x = DoTest(lbuf(0, o) , hldtab(0, 0) , testdef(0, 0) , results(0, 0) , events, ode, lasttst) For y = 0 To 1023 If results (2, y) <> 0 Then gridi.Row = gridl.Row + 1 gridl.Rol = 0 gridl.Text = "bead " & y gridl.Rol = 1 gridl.Text = results(2, y) End If SUBSTITUTE SHEET (RULE 26) Next y End Sub Sub TCInit Click () Dim x As Integer Dim y As Integer 'x = InitBrd() If AssaySel.Text Then Oplnst.Text = "You must select a Cube and Assay first!"
Else For x = 0 To 1023 For y = 0 To 3 results(y, x) = 0 Next y Next x Oplnst.Text - "Initialization Complete"
TCStart.Enabled = True TCHa1t.Enabled = True End If End Sub Sub TCStart Click () Dim x As Integer Dim y As Integer Dim z As Integer Dim measure As Integer x = DoTest(lbuf(0, 0), hldtab(0, 0), testdef(0, 0), results(0, 0), events ode, lasttst) dataint.Refresh Do While Not dataint. Records et. EOF
If AssaySel.Text = dataint.Recordset("assay") Then For x = 0 To 4 resline(x) = dataint.Recordset(x + 2) 'move to temp area Next x z = resline(0) 'token value results (2, z) = results(2, z) + 1 results (0, z) = results(0, z) + 1 If resline(2) = 0 Then measure = results (3, z) / results (2, z) 'sum green over total Else measure = results(1, z) / results(0, z) over count divided k End If If ((measure >= resline(3)) And (measure <= resline(4))) Then gridl.Row = gridl.Row + 1 gridl.Col = 0 gridl.Text = tkname(z) gridl.Rol = 1 gridl.Text = data:Lnt. Records et ("interpretation") End If SUBSTITUTE SHEET (RULE 26) End If dataint.Recordset.MoveNext Loop graphl.RandomData = 1 graphs. Refresh state = 0 'AssaySel.Clear Oplnst.Text = "Test Complete"
End Sub Sub Timerl Timer O
Dim x, y, z As Integer y = ReadPanel(tbuf(0)) x = tbuf(38) ' set Waste Water indicator y = x And 1 z = 1 If y = 1 Then z = 0 WWPic(z) Visible = False WWPic(y).Visible = True ' set Sheath Fluid y = (x And 2) / 2 z = 1 If y = 1 Then z = 0 SFPic(z).Visible = False SFPic(y).Visible = True 'set pressure y = (x And &H80) / &H80 z = 1 If y = 1 Then z = 0 PRPic(z).Visible = False PRPic(y).Visible = True 'set flow y = x And &H70 'mask bits Select Case y Case &H10 FCPic(0).Visible = True FCPic(1).Visible = False FCPic(2).Visible = False Case &H20 FCPic(0).Visible = False FCPic(1).Visible = False FCPic(2).Visible = True Case &H40 FCPic(z).Visible = False FCPic(1).Visible = True FCPic(2).Visible = False Case 0 FCPic(0).Visible = False FCPic(1).Visible = False SUBSTITUTE SHEET (RULE 26) FCPic(2). Visible = True End Select End Sub SUBSTITUTE SHEET (RULE 26) Option Explicit Dim photo(5) As Integer Dim ampnum(5) As Integer Dim mode(s) As Integer Dim thresh(5) As Integer Dim f comp (5) As Integer Dim trigsav As Integer Sub Check3D1_Click (Value As Integer) pbuf (39) = Value End Sub Sub Check3D2_Click (Value As Integer) pbuf (40) = 2 * (Value + 1) End Sub Sub setpvals ( ) this sets the manual adjust screen to reflect the current values in pbuf Dim x As Integer For x = 0 To 4 txtNumber(x) = pbuf(x + 13) amp (x) = pbuf (x + 18) Next x fscmod(pbuf (23)).Value = True sscmod(pbuf (24)) Value = True fllmod(pbuf (25)) Value = True fl2mod(pbuf(26)).Value = True fl3mod(pbuf (27) ) Value = True For x = 29 To 33 If pbuf(x) <> 0 Then trigval = pbuf(x) trigger(x - 29) Value = True End If Next x For x = 0 To 3 textl (x) = pbuf (x + 34) Next x ddmtxt(0) = pbuf(41) ddmtxt(1) = pbuf(42) If pbuf(40) <> 0 Then optionl(pbuf(40) - 2).Value = True stat(0).Caption = Format$ (pbuf (10) * .05, "###.00") stat(l).Caption = Format$(pbuf(11) * .02, "###.00") stat(2).Caption = Format$(pbuf(12) / 100, "#''##.00") If pbuf (3 9) = 1 Then stat(l).Caption = "Enabled"
Else stat(3).Caption = "Disabled"
End If SUBSTITUTE SHEET (RULE 26) End Sub Sub amp_Change (Index As Integer) if Val(amp(Index).Text) > 999 Then amp(Index).Text = "999"
pbuf(Index + 18) = Val(amp(Index).Text) amp(Index).Text = Format (pbuf (Index + 18)) End sub Sub Command3Dl_Click () form2.Hide End Sub Sub Command3D2 Click () Dim x As Integer Dim y As Integer Dim z As Integer y = ReadPanel(tbuf(O)) For x - 13 To 42 If pbuf(x) <> tbuf(x) Then y = SendPanel(x, pbuf(x)) Next x End Sub Sub Command3D3 Click () Dim y As Integer Dim x As Integer loadpbuf setpvals For x = 13 To 42 y - SendPanel(x, pbuf(x)) If y > 30 Then MsgBox "Flow Cytometer is not responding", 48, "Set Flow Cy r"
Next x End Sub Sub Command3D4_Click () savepbuf End Sub Sub Command3D5_Click () form2.Hide End Sub Sub ddmscroll Change (Index As Integer) pbuf(Index + 41) = ddmscroll(Index).Value ddmtxt(Index) = Format (ddmscroll (Index)) End Sub Sub ddmtxt Change (Index As Integer) If Val(ddmtxt(Index)) > 999 Then ddmtxt(Index) _ "999"
ddmscroll(Index).Value = Val (ddmtxt (Index)) pbuf(Index + 41) = ddmscroll(Index).Value End Sub SUBSTITUTE SHEET (RULE 26) sub Sub flimod Click (Index As Integer, Value As Integer) pbuf(25) = Index If Index = 0 Then spina(2).Enabled = False amp(2).Enabled = False Else spina(2).Enabled = True amp(2).Enabled = True End If End Sub Sub fl2modClick (Index As Integer, Value As Integer) pbuf(26) ---7-Index If Index = 0 Then spina(3).Enabled = False amp(3).Enabled = False Else spina(3).Enabled = True amp (3) . Enabled = True End if End Sub Sub fl3mod Click (Index As Integer, Value As Integer) pbuf(27) = Index If Index = 0 Then spina(4).Enabled = False amp(4).Enabled = False Else spina(4).Enabled = True amp(4).Enabled = True End If End Sub Sub Form Load ) Dim x As -Integer Dim y As Integer loadpbuf setpvals form2.Caption = forml!CubeSel.Text & " Cube Manual Adjust"
End Sub Sub fscmod Click (Index As Integer, Value As Integer) pbuf(23) = Index If Index = 0 Then spina(o).Enabled = False amp(0).Enabled = False Else spina(0).Enabled = True amp(O).Enabled = True End If End Sub Sub HScroil2_Change (Index As Integer) SUBSTCTUTE SHEET (RULE 26) pbuf(Index + 34) = HScrolli(Index).Value textl(Index) = Format(HScrolll(Index)) End Sub Sub optionl Click (Index As integer) pbuf(40) = Index + 2 pbuf(39) = 1 End Sub Sub Spina_SpinDown (Index As Integer) Dim min pbuf(Index + 13) = pbuf(Index + 13) - 1 If Index = 0 Then min = 0 Else min = 150 If pbuf(Index + 13) < min Then pbuf(Index + 13) = min txtNu- er(Index).Text Format(pbuf(Index + 13)) End Sub Sub Spini_SpinUp (Index As Integer) Dim max pbuf(Index + 13) = pbuf(Index + 13) + 1 If Index = 0 Then max = 4 Else max = 999 If pbuf(Index + 13) > max Then pbuf (Index + 13) = max txtNumber(Index).Text = Format(pbuf(Index + 13)) End Sub Sub Spina_SpinDown (Index As Integer) Dim min pbuf (Index, + 18) = pbuf(Index + 18) - 1 min = 100 If pbuf(Index + 18) < min Then pbuf (Index + 18) = min amp(Index).Text = Format(pbuf(Index + 18)) End Sub Sub Spina_SpinUp (Index As Integer) Dim max pbuf(Index + 18) = pbuf(Index + 18) + 1 max = 999 If pbuf (Index + 18) > max Then pbuf (Index + 18) = max amp (Index). Text = Format (pbuf (Index + 18)) End Sub Sub sscmod Click (Index As Integer, Value As Integer) pbuf(24) = Index If Index = 0 Then spina(1).Enabled = False amp(1).Enabled = False Else spina(1).Enabled = True amp(1).Enabled = True End If End Sub Sub Textl_Change (Index As Integer) If Val(textl(Index)) > 999 Then textl(Index) _ "999"
HScrolll(Index).Value = Val (textl (Index)) pbuf (Index + 34) = HScrolll (Index) Value SUBSTITUTE SHEET (RULE 26) End Sub Sub trigger_Click (Index As Integer, Value As Integer) Dim x For x = 29 To 33 pbuf(x) = 0 Next x pbuf(Index + 29) = Val(trigval.Text) trigsav = Index pbuf (28) = 128 + Index End Sub Sub trigvalChange If Val(trig_al.Text) O
> 999 Then trigval.Text = 11999"
VScrolll.Value = Val(trigval.Text) pbuf(trigsav + 29) = VScrolll.Value End Sub Sub VScrolll_Change () trigval.Text = Format(VScrolll.Value) pbuf(trigsav + 29) = VScrolll.Value End Sub SUBSTITUTE SHEET (RULE 26) option Explicit Global pbuf(64) As Integer Global tbuf(64) As Integer Global lbuf(8, 32) As Integer Global hldtab(7, 1023) As Integer Global results(3, 1023) As Long Global testdef(1, 1023) As Integer Global resline(5) As Integer Global tkname(1024) As String Global state As Integer Global lastnode As Integer Global lasttst As Integer Global events As Long Declare Function DoTest Lib "c:\msvc\bin\orbit.dll" (lbuf As Integer, hldt Integer, testdef As Integer, results As Long, ByVal events&, ByVal lastnod Val lasttst%) As Integer Declare Function InitBrd Lib "c:\msvc\bin\orbit. dll" () As Integer Declare Function SendPanel Lib "c:\msvc\bin\orbit. dlI" (ByVal parm%, ByVal 11) As Integer Declare Function ReadPanel Lib "c:\msvc\bin\orbit.dll" (pbuf As Integer) A
ger Declare Function ReadList Lib "c:\msvc\bin\orbit.dll" (ibuf As Integer) As er Sub loadpbuf ( ) ' for a selected cube, the data base values are loaded into pbuf Dim x As Integer form2lfacset.Refresh Do While form2!facset.Recordset.EOF = False If forml!CubeSel.Text = form2!facset.Recordset(0) Then Exit Do form2!facset.Recordset.MoveNext Loop For x = 0 To 42 pbuf(x) = form2!facset.Recordset(x + 1) Next x End Sub Sub savepbuf () This saves the current values in pbuf to the data base Dim x As Integer form2! facset.Refresh Do While form2lfacset.Recordset.EOF = False If formllCubeSel.Text = form2!facset.Recordset(0) Then Exit Do form2!facset.Recordset.MoveNext Loop form2lfacset.Recordset.Edit For x = 0 To 42 form2lfacset.Recordset(x + 1) = pbuf(x) Next x form21facset.Recordset.Update End Sub SUBSTITUTE SHEET (RULE 26) Option Explicit SUBSTITUTE SHEET (RULE 26) HDXDLL.DLL source.
//

#include <windows.h>
#include "mdxdll.h"

int WINAPI _export InitBrd(void) {
rasm {
mov dx,brdctrl mov ax,20h out dx,ax mov dx,brdctrl in ax,dx or ax,outfifoclr ;set both fifos to clear or ax,inifoclr out dx,ax mov dx,inpxfer mov ax,O ;set xfer count to 0 out dx,ax mov dx,prtctrl mov ax,0 or ax,ctlO ;set control lines to idle or ax,ctll out dx,ax mov dx,prtctrl in ax,dx mov bx,preset ;reset face not bx and ax,dx out dx,ax or ax,preset ;toggle out dx,ax mov dx,brdsts in ax, dx:

mov dx,inpxfer mov ax,O
out dx,ax mov dx,prtctrl may ax,6 ;assert ctlO &l out dx,ax // asm code here ) //SendPanel(ddmer a,l);
return(0);

int WINAPI _export SendPanel(int parm, int PanVal) SUBSTITUTE SHEET (RULE 26) Lnt i = 0;

asm w {
mov dx,brdctrl in ax,dx mov bx,inen not bx and ax,bx or ax,outen out dx,ax mov dx,prtctrl in ax,dx mov bx,ctlO
not bx and ax,bx out dx,ax mov dx,brdctrl in ax,dx or ax,outfifoclr out dx,ax mov bx,outfifoclr not bx and ax,bx out dx,ax mov dx,bdata mov ax,l out dx,ax mov bx,parm ;get code shl bx,10 mov ax,PanVal ;get panel value and ax,3ffh or ax,bx out dx,ax ;send it out mov dx,dlyctrl mov ax,0160h out dx,ax mov dx,brdctrl in ax,dx or ax,hshken out dx,ax mov cx,4000 wsend: mov dx,brdsts in ax,dx mov bx,ax and bx,l ;1=empty fifo loope wsend and ax,40h ;bit 6 = 1 = xfer pending jnz wsend mov dx,prtctrl in ax,dx or ax,ctlO
out dx,ax SUBSTITUTE SHEET (RULE 26) mov dx,brdctrl -186-in ax,dx mov bx,outen or bx,hshken not bx and ax,bx out dx,ax mov dx,dlyctrl mov ax,OlOlh out dx,ax // asm code here }
return(i);
}

int. WINAPI _export ReadPanel(int _far *pbuf) {
int far* pbufptr = pbuf;
_asm {
may dx,brdctrl in ax,dx or ax,outfifoclr ;set both fifos to clear or ax,infifoclr out dx,ax mov dx,inpxfer mov ax,O ;set xfer count to 0 out dx,ax may dx,prtctrl mov ax,0 or ax,ctl0 ;set control lines to idle or ax,ctll out dx,ax mov dx,prtctrl in ax,dx or ax,preset ;toggle out dx,ax lowlp: mov dx,prtsts in ax,dx mov bx,ax and ax,stsl ;has stsl gone low yet?
jz waithigh ;yes, wait for it to go back high imp lowlp ;******,r,r*******

waithigh:
mov dx,brdctrl ;no, panel coming so set up read in ax,dx and al,Oeeh ;handsk off out off or ax,8 ;clr in fifo out dx,ax SUBSTITUTE SHEET (RULE 26) and a1,Ut7h ;fiPo ctr ott out dx,ax mov dx,inpxfer ;set up xfer count may ax,43 out dx,ax may dx,prtctrl in ax,dx may bx,ctlO
not bx and ax,bx out dx,ax ;set ct10 to request panel data may dx,dlyctrl mov ax,101h out dx,ax may dx,brtctrl in ax,dx or ax,hahken ;initiate handshake out dx,ax waitdata:
may dx,brdsts may bx,xferincom or bx,xferpend in ax,dx and ax,bx ;wait until xfer not pending jnz waitdata ;or incomplete mov dx,prtctrl ;done in ax,dx or ax,ctl0 ;stop out dx,ax mov dx,brdctrl in ax,dx and al,Oebh ;turn off handshake and input enable out dx,ax push di push es mov cx,43 lea di,pbufptr mov dx, bdata readlp:
in ax,dx ;read data from fifo mov bx,ax and ax,3ffh and bx,OfcOOh shr bx,9 ;right 10 * 2 mov word ptr es:[di.+bxl,ax loop readlp pop es pop di }
return(0);
}

int WINAPI _export DoTest(int _far *lbuf, int _far *hldtab, int _far *testdef, long __far *results, long events, int lastnode, int lasttst) SUBSTITUTE SHEET (RULE 26) { .188-int far* lbufptr = lbuf;
tot far* hldptr = hidtab;
int far* testptr - testdef;
long far* resptr - results;

int x,y,z,bdi,goodcnt,token = 0;
while (events > 0) {
goodcnt - ReadList(lbufptr);
if (goodcnt > 0) {
for (bdi 0; bdi < goodcnt; bdi++) {
xy=0;
// orange correction goes here for lbufptr[bdi)[f12]
// x is the current node in the hid table token = -1;
while (token < 0) {
z - lbufptr(bdi * 8 + (hldptr[x * 8))];
// z is the value of the parameter under test if ((z >= hidptr(x * 8 + 2)) && (z <= hldptr[x * 8 + 3])) val. && high val {
if (hldptr[x * 8 + 4] =- 0) token - hldptr[x * 8 / 0 means done, get token true else x = hldptr(x * 8 + 4]; /1 get node true }
else {
if (hldptr(x * 8 + 5] 0) token - hidptr[x * 8 / get token false also x = hidptr[x * 8 + 5]; // get node false }
}
events--,-z - lbufptr(bdi * 8 + 2]; // z is FL1 if (z < testptr(token * 2 + 1]) resptr[token * 41++; //ir count if (z > testptr[token * 2 + 1]) resptr(token * 4 + 1]++;
ver count resptr[token * 4 + 21++; // inc total count rezptr(token * 4 + 3] += z; //sum FL1 }
}

}
_asm nap }

return(0);
}

int WINAPI _export ReadList(int _far *lbuf) SUBSTITUTE SHEET (RULE 26) c int far* lbufptr lbuf;
int 1 0;

asm {

read list mode *************************************************************
mov dx,brdctrl in ax,dx or ax,outfifoclr ;set both fifos to clear or ax,infifoclr out dx,ax mov dx,inpxfer mov ax,O ;set xfer count to 0 out dx,ax mov dx,prtctrl mov ax,O
or ax,ctlO ;set control lines to idle or ax,ctll out dx,ax may dx,prtctrl in ax,dx or ax,preset ;toggle .out dx,ax lowlpl: mov dx,prtsts in ax,dx mov bx,ax and ax,stsO ;has stsO gone low yet?
jz waithighi ;yes, wait for it to go back high jmp lowlpl ; ********

waithighi:
mov dx,brdctrl ;no, list mode coming so set up read in ax,dx and al,Oeeh ;handsk off out off or ax, 8 ;clr in fifo out dx, ax and al,Of7h ;fifo clr off out dx,ax push es push di push ds push si les di,lbufptr mov dx,inpxfer ;set up xfer count may ax,120 ;7 vale + chk sum * 15 events out dx,ax may dx,prtctrl in ax,dx SUBSTM fE SHEET (RULE 26) may ox,ctll -190-not bx and ax,bx out dx,ax ;set ctll to request list data mov dx,dlyctrl mov ax,lOlh out dx,ax mov dx,brdctrl in ax,dx or ax,hshken ;initiate handshake out dx,ax waitdatal:
mov dx,brdsts mov bx,xferincom or bx,xferpend in aLx, dx and ax,bx ;wait until xfer not pending jnz waitdatal ;or incomplete mov dx,bdata mov cx,120 ;number of words to read chunk: in ax,dx stosw loop chunk mov dx,prtctrl ;done in ax,dx or ax,ctll ;stop out dx,ax mov dx,brdctrl in ax,dx and al,Oebh ;turn off handshake and input enable out dx,ax les di,lbufptr lds si, lbufptr mov cx,15 ;up to 15 good events could be present ;check alignment mov dx,O ;count of good records test word ptr (si),Oe000h jz alnok dec cx ;only 14 max possible now push cx mov cx,7 ;seven other possible alignments alnlp: lodsw test ax,Oe000h jz nowalnok ;now have alignment loop alnlp pop cx jmp badbuff nowalnok:
sub si,2 ;repoint to good align pop cx ;restore loop count alnok: push cx mov cx,7 mov bx,O
sumchk: lodsw SUBSTITUTE SHEET (RULE 26) add bx, ax -191-loop sumchk lodsw cmp bx,ax ;ie check sum good jne nocopy sub si,16 ;repoint to start of rec mov cx,8 clncpy: lodsw and ax,3ffh ;knock off parm number stosw loop clncpy inc dx ;count good records nocopy: pop cx loop alnok badbuff:
mov i,dx pop si pop ds pop di pop es }
return(i);
}

II
DLL initialization and exit.

int WINAPI LibMain(HANDLE hinst, WORD wDataSeg, WORD cbHeapSize, LPSTR lpszCmdLine) {
hNodinst - hInst;
if (cbHeapsize 1= 0) UnlockData(O);
return 1;
}
int _export WINAPI WEP(int nParam) {
return 1;
}

SUBSTITUTE SHEET (RULE 26) /f Include file for l4DXDLL.DLL
//
I/////Ill/I//III///f111///!/1//I/I/III//f/II//11111/1111!/I
#ifdef _cplusplus extern "C" {
#endif Define DEBUG macros.

*-if defined( DEBUG) && /defined( AFX) char _az ASSERT[255);

#define ASSERT(a) if(1(a)) { wsprintf(_sz ASSERT, \
"assertion failed in file %s at line %d\\r\n", \
(LPSTR)(_FILE_), LINE ); OutputDebugString(^sz ASSERT); }
#define TRACE(a) OutputDsbugString(a"\r\n") #define TRACEI(a,b) { wsprintf(_sz ASSERT(a"\r\n",(int)(b)); \
OutputDebugString(_sz ASSERT); }
#define TRACE2(a,b,c) { wsprintf(_sz_ASSERT, a"\r\n",(int)(b),(int)(c)); \
OutputDebugString(_sz ASSERT)] }

#endif #if !defined( DEBUG) && /defined( AFX) #define ASSERT(a) #define TRACE(a) #define TRACEI(a,b) rdefine TRACE2(a,b,c) #endif III//I/III//I////III//III/II!/III//I//I//II///I//II/II/I/I/
From Windowsx.h II
#define GlobalPtrHandle(lp) \
((HGLOBAL)LOWORD(GlobalHandle(SELECTOROF(lp)))) #define GlobalLockPtr(lp) \
((BOOL)SELECTOROF(GlobalLock(GlobalPtrHandle(lp)))) 'define GlobalUnlockPtr(lp) GlobalUnlock(GlobalPtrHandle(lp)) Odefine GlobalAllocPtr(flags, cb) \
(GlobalLock(GlobalAlloc((flags), (cb)))) 'define GlobalReAllocPtr(lp, cbNew, flags) \
(GlobalUnlockPtr(lp), GlobalLock(Glob alReAlloc(Glob alPtrHandle(lp) , (cb lags)))) 'define GlobalFreePtr(lp) (GlobalUnlockPtr(lp), (BOOL)Glob alFree(Glob alPtrHandle(lp))) SUBSTITUTE SHEET (RULE 26) Global variables.

HINSTANCE hModlnst; // module handle.
// Cytomation board definitions :define base 0x240 define bdata base + 0 #define brdctrl base + 2 #define brdsts base + 4 #define prtctrl base + 6 #define prtsts base + 8 :define dlyctrl base + OxOa #defins inpxfer base +.OxOc //facs codes #define ddmena 39 //board control #define outen 1 #define outfifoclr 2 #define men 4 #define infifoclr 8 #define hshken Ox10 //board status #define outfifoe 1 #define outfifoh 2 #define outfifof 4 ;define infifoe 8 #define infifoh Ox10 ;define infifof Ox20 :define xferpend Ox4O
#define xferincom 0x80 //port control #define preset 1 define ctlO 2 #define ctll 4 //port status :define eir 1 :define pats 2 :define stsO 4 ;define stsl 8 II
Exported Functions.

SUBSTITUTE SHEET (RULE 26) // Local Functions.

int WINAPI _export SandPanel(int parm, int PanVal);
int WINAPI _export ReadPanel(int pbuf[]);
int. WINAPI _export ReadList(int lbuf[]);
int WINAPI _export InitBrd (void);
int. WINAPI _export 1)oTest(int lbuf[], int hldtab[], int testdef[j, long results[), long events, int lastnode, int lasttst);
ifdef _cplusplus }
Oendif SUBSTITUTE SHEET (RULE 26) ----------------------------------------------------------Module Definition file for MDXDLL.DLL
----------------------------------------------------------LIBRARY MDXDLL

DESCRIPTION 'MDXDLL Library' EXETYPE WINDOWS

CODE PRELOAD MOVABLE
DATA PRELOAD MOVABLE

EXPORTS
WEP @1 RESIDENTNAME
InitErd @2 SendPanel @3 ReadPanel @4 ReadList @5 DoTest @6 SUBSTITUTE SHEET (RULE 26)

Claims (59)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. A method of preparing a beadset capable of detecting a plurality of analytes in a single fluid sample by flow cytometric analysis comprising:
(a) obtaining a plurality of subsets of beads wherein the beads in each subset are sufficiently homogeneous with respect to at least three selected classification parameters (C1, C2, C3 ... C n) and sufficiently different in at least one of said classification parameters from beads in any other subset so that a profile of classification parameter values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameters does not include a number of the beads in said each subset;
(b) coupling the beads within each subset to a reactant that will specifically react with a given analyte of interest in a fluid to be tested; and (c) mixing the subsets of beads to produce a beadset, wherein the subset identity and therefore the reactant to which the beads has been coupled is identifiable by said flow cytometry based on the profile of the beads.
2. A beadset capable of detecting a plurality of analytes in a single fluid sample by flow cytometric analysis comprising a plurality of subsets of beads wherein:
(a) the beads in each subset are sufficiently homogeneous with respect to at least three selected classification parameters (C1, C2, 3... C n) and sufficiently different in at least one of said classification parameters from beads in any other subset so that a profile of classification parameter values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameters does not include a number of the beads in said each subset;
(b) wherein the beads within each subset are coupled to a reactant that will specifically react with a given analyte of interest in a fluid to be tested;
and (c) wherein said subsets have been mixed to produce the beadset, characterized in that the subject identity and therefore the reactant to which the beads has been coupled is identifiable based on the profile of the beads.
3. A method of flow cytometric analysis capable of detecting a plurality of analytes of interest in a single fluid sample comprising:
(a) obtaining a beadset comprising a plurality of subsets of beads wherein the beads in each subset;
(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3 ... C n) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that a profile of the classification parameter values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameter values does not include a number of the beads in said each subset; and (2) are coupled to a reactant that will specifically react with a selected analyte of interest in a fluid to be tested;
(b) mixing, to produce a reacted bead sample, the beadset with the fluid under conditions that will allow reactions between analytes of interest in the fluid and the reactants on the beads in said beadset, wherein the reactions cause a change in a value of a fluorescent signal (F m) emitted from said bead;
(c) analyzing the reacted bead sample by said flow cytometry to determine the profile and the F m value of each bead analyzed;
(d) identifying the subject to which each bead belongs and therefore the reactant on the bead as a function of the profile of the classification parameter values;
and (e) detecting the presence or absence of a particular analyte of interest in said fluid as a function of the identification in step (d) and the F m values.
4. A method of flow cytometric analysis capable of detecting a plurality of analytes of interest in a single fluid sample comprising:

(a) obtaining a beadset comprising a plurality of subsets of beads wherein the beads in each subset;

(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3 ... C n) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that a profile of the classification parameter values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameter values does not include a number of the beads in said each subset; and (2) are coupled to a reactant that will specifically react with a selected analyte of interest in a fluid to be tested;
(b) mixing, to produce a reacted bead sample, the beadset with the fluid under conditions that will allow reactions between analytes of interest in the fluid and the reactants on the beads in said beadset;

(c) mixing with the reacted bead sample a fluorescent label under conditions such that said label will bind to and thereby increase a value of a fluorescent signal F,, emitted from said beads;

(d) analyzing the reacted bead sample containing the fluorescent label by said flow cytometry to determine the profile and the F m value of each bead analyzed;
(e) identifying the subset to which each bead belongs and therefore the reactant on the bead as a function of the profile of the classification parameter values;
and (f) detecting the presence or absence of a particular analyte of interest in said fluid as a function of the identification in step (e) and the F m values.
5. A method of flow cytometric analysis capable of detecting a plurality of analytes of interest in a single sample comprising:

(a) obtaining a beadset comprising a plurality of subsets of beads wherein the beads in each subset;

(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3 ... C n) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that a profile of the classification parameter values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameter values does not include a number of the beads in said each subset, (2) are coupled to a reactant that will specifically react with a selected analyte of interest in a fluid to be tested, and (3) are reacted with a fluorescently labelled compound which competes with said selected analyte of interest for reaction with said reactant;
(b) mixing, to produce a reacted bead sample, the beadset with the fluid under conditions that will allow reactions between analytes of interest in the fluid and the reactants on the beads in said beadset and thereby to allow the analytes of interest to competitively inhibit or displace the fluorescently labelled compounds from said beads, resulting in a decrease in a fluorescent signal F m emitted from a bead with which an analyte of interest in the fluid has reacted;

(c) analyzing the reacted bead sample by said flow cytometry to determine the profile and an F m value of each bead analyzed;
(d) identifying the subset to which each bead belongs and therefore the reactant on the bead as a function of the profile of classification parameter values;
and (e) detecting the presence or absence of a particular analyte of interest in said fluid as a function of the identification in step (d) and the F m values.
6. The method of any one of claims 3, 4 and 5 wherein C1, C2, and C3 are each different and are selected from the group consisting of forward light scatter, side light scatter and fluorescence.
7. The method of any one of claims 3, 4 and 5 wherein n is greater than or equal to 4 C1 is forward angle light scatter, C2 is side angle light scatter, C3 is fluorescence at a first wavelength and C4 is fluorescence at a second wavelength.
8. The method of claim 7 wherein said first wavelength is red and said second wavelength is orange.
9. The method of claim 7 wherein said first wavelength is red, said second wavelength is orange, and the wavelength of said fluorescent signal F m is green.
10. The method of claim 3 wherein said analytes of interest are antigens and said reactants are antibodies specifically reactive with said antigens.
11. The method of claim 3 wherein said analytes of interest are antibodies and said reactants are antigens specifically reactive with said antibodies.
12. The method of claim 3 wherein said analytes of interest are antigens selected from the group consisting of bacterial, viral, fungal, mycoplasmal, rickettsial, chlamydial and protozoal antigens and said reactants are antibodies specifically reactive with said antigens.
13. The method of claim 3 wherein said reactants are antigens selected from the group consisting of bacterial, viral, fungal, mycoplasmal, rickettsial, chlamydial and protozoal antigens and said analytes of interest are antibodies specifically reactive with said antigens.
14. The method of any one of claims 10 or 12 wherein said antigens are antigens borne by pathogenic agents responsible for sexually transmitted disease.
15. The method of any of claims 10 or 12 wherein said antigens are antigens borne by pathogenic agents responsible for a pulmonary disorder.
16. The method of any of claims 10 or 12 wherein said antigens are antigens borne by pathogenic agents responsible for a gastrointestinal disorder.
17. The method of claim 3 wherein said analytes of interest are substances of abuse.
18. The method of claim 3 wherein said analytes of interest are therapeutic drugs.
19. The method of claim 3 wherein said analytes of interest are antigens or antibodies associated with one or more selected pathological syndromes.
20. The method of claim 19 wherein said syndromes are selected from the group consisting of malignancy, allergy, autoimmune diseases, and blood borne viruses.
21. The method of claim 19 wherein at least one of said syndromes is a cardiovascular disorder.
22. The method of claim 3 wherein said analytes of interest are selected from the group consisting of analytes testing for pregnancies and hormones.
23. The method of claim 3 wherein said fluorescent signal is emitted from fluoresceinated antibodies specific for antigens coupled to said beads in said beadset.
24. The method of claim 3 wherein said fluorescent signal is emitted from a fluoresceinated compound specifically reactive with an immunoglobulin molecule.
25. The method of claim 3 wherein said fluorescent signal is emitted from an agent selected from the group consisting of a fluoresceinated anti-immunoglobulin antibody or a specifically reactive fragment thereof, fluoresceinated protein A, and fluoresceinated protein G.
26. The method of claim 5 wherein said analytes of interest comprise autoantibodies, said reactants comprise oligopeptide epitopes reactive with said autoantibodies, said fluorescently labelled compounds comprise fluorescent monoclonal antibodies reactive with said epitopes, and wherein the presence of the autoantibodies is detected as a result in a decrease of F..
27. The method of claim 3 wherein said analytes of interest are enzymes, said reactants are fluorescently labelled substrates for said enzymes, and a change in F.
results from cleavage of said substrates from said beads.
28. The method of claim 27 wherein said enzymes are selected from the group consisting of proteases, glycosidases, nucleotidases, oxidoreductases, hydrolyases, esterases, convertases, ligases, transferases, phosphyorylases, lyases, lipases, peptidases, dehydrogenases, oxidases, phospholipases, decarboxylases, invertases, aldolases, transaminases, synthetases, and phosphatases.
29. The method of claim 3 wherein the fluid is selected from the group consisting of plasma, serum, tears, mucus, saliva, urine, pleural fluid, spinal fluid, gastric fluid, sweat, semen, vaginal secretions, fluid from ulcers and other surface eruptions, blisters, and abscesses, and extracts of tissues including biopsies of normal, malignant, and suspect tissues.
30. A method of flow cytometric analysis for detection of immunoglobulins in a fluid sample comprising the steps of:
(a) obtaining a beadset comprising a plurality of subsets of beads wherein the beads in each subset;
(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (Cl, Ca C3 ... C) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that a profile of the classification parameter values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameter values does not include a number of the beads in said each subset; and (2) are coupled to an immunoglobulin that corresponds to one of the immunoglobulins to be detected in the fluid sample;
(b) obtaining a fluorescently labelled immunoglobulin-binding reagent capable of reacting with the immunoglobulins to be detected;
(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample and the fluorescently labelled immunoglobulin-binding reagent under conditions that will allow competitive binding reactions between the immunoglobulin-binding reagent and immunoglobulin in the fluid sample and between the immunoglobulin-binding reagent and the immunoglobulin on the beads, wherein a reaction between a bead-bound immunoglobulin and the fluorescently labelled immunoglobulin-binding reagent causes an increase in the value of a fluorescent signal (F m) emitted from said bead;
(d) analyzing the reacted bead sample by said flow cytometry to determine the profile and an F m value of each bead analyzed;
(e) identifying the subset to which each bead belongs and therefore the immunoglobulin on the bead as a function of the profile of the classification parameter values; and (f) detecting immunoglobulins in said fluid sample as a function of the identification in step (e) and the F m values.
31. The method of claim 30 wherein said immunoglobulins to be detected are immunoglobulins belonging to different immunoglobulin classes.
32. The method of claim 31 wherein said classes are selected from the group consisting of IgG, IgM, IgA, and IgE.
33. The method of claim 30 wherein said immunoglobulins to be detected are immunoglobulins belonging to different immunoglobulin sub-classes.
34. The method of claim 33 wherein said sub-classes are selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
35. A method of flow cytometric analysis for detection of immunoglobulin specific for a particular epitope of interest in a sample comprising the steps of:
(a) obtaining a beadset comprising a plurality of subsets of beads wherein the beads in each subset;
(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3 ... C n) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that a profile of the classification parameter values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameter values does not include a number of the beads in said each subset; and (2) are coupled to a monoclonal antibody preparation which is specific for an epitope that binds to an immunoglobulin to be detected;
(b) obtaining a plurality of fluorescently labelled reagents wherein each of said reagents bears an epitope that binds to the monoclonal antibody preparation coupled to the beads within a subset;
(c) mixing, to produce a reacted bead sample, the beadset with the sample and the fluorescently labelled reagents under conditions that will allow competitive binding reactions between the fluorescently labelled reagents and immunoglobulin in the sample and between the fluorescently labelled reagents and the monoclonal antibody preparations on the beads wherein a reaction between a bead-bound antibody and the fluorescetly labelled reagent causes an increase in a value of a fluorescent signal (Fm) emitted from said bead;

(d) analyzing the reacted bead sample by said flow cytometry to determine the profile and the F m value of each bead analyzed;

(e) identifying the subset to which each bead belongs and therefore the monoclonal antibody preparation on the bead as a function of the profile of the classification parameter values; and (f) detecting the presence or absence of an immunoglobulin in said sample specific for said particular epitope as a function of the identification in step (e) and the F. values.
36. The method of claim 35 wherein the particular epitope is an epitope located on a viral antigen.
37. The method of claim 36 wherein said viral antigen is an antigen from HIV.
38. A method of flow cytometric analysis for detection of analytes commonly elevated in pregnancy in a fluid sample comprising the steps of:
(a) obtaining a beadset comprising a plurality of subsets of beads wherein the beads in each subset;
(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3 ... C n) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that a profile of the classification parameter values within each subject detectable by flow cytometry is unique, wherein the at least one of said classification parameter values does not include a number of the beads in said each subset; and (2) are coupled to an antibody which is specific for an analyte to be detected in the fluid sample;
(b) obtaining a plurality of preparations of antibody molecules wherein each of said preparations contains fluorescently labelled antibodies specific for an analyte to be detected in the fluid sample;

(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample and the fluorescently labelled antibodies under conditions that will allow binding reactions between the antibodies that are coupled to the beads, the analytes, and the fluorescently labelled antibodies so as to bind said fluorescently labelled antibodies to said beads through binding to said analytes which are in turn bound to said beads through said bead-bound antibodies and wherein a bridging reaction between a bead-bound antibody, the analyte to which that antibody binds, and the fluorescently labelled antibody specific for said analyte causes an increase in a value of a fluorescent signal (F m) emitted from said bead;

(d) analyzing the reacted bead sample by said flow cytometry to determine the profile and the F m value of each bead analyzed;
(e) identifying the subset to which each bead belongs and therefore the antibody on the bead as a function of the profile of the classification parameter values;
and (f) detecting the analytes in said fluid sample as a function of the identification in step (e) and the F m values.
39. The method of claim 38 wherein said analytes are selected from the group consisting of human chorionic gonadotropin, alpha fetoprotein, and 3' estradiol.
40. A method of flow cytometric analysis for determining an epitope to which a monoclonal antibody binds comprising the steps of:
(a) obtaining a beadset comprising a plurality of subsets of beads wherein the beads in each subset;
(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3 ... C n) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that a profile of the classification parameter values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameter values does not include a number of the beads in said each subset; and (2) are coupled to a peptide which provides a given epitope;
(b) obtaining a fluorescently labelled monoclonal antibody of interest;

(c) mixing, to produce a reacted bead sample, the beadset with the fluorescently labelled monoclonal antibody under conditions that will allow binding reactions between the bead-bound peptide which provides the epitope to which the monoclonal antibody is capable of binding and said monoclonal antibody, wherein the binding reactions cause an increase in a value of a fluorescent signal (F m) emitted from said beads;

(d) analyzing the reaction bead sample by said flow cytometry to determine the profile and the F m value of each bead analyzed;

(e) identifying the subset to which each bead belongs and therefore the peptide on said bead as a function of the profile of the classification parameter values;

and (f) detecting the epitope to which the monoclonal antibody binds as a function of the identification in step (e) and the F m values.
41. The method of claim 40 where said peptides are from 2-100 amino acids in length.
42. A method of flow cytometric assay for antibodies reactive with pathogens of interest in a fluid sample comprising the steps of:
(a) obtaining a beadset comprising a plurality of subsets of beads wherein the beads in each subset;

(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3 ... C n) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that a profile of the classification parameters values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameter values does not include a number of the beads in said each subset; and (2) are coupled to an antigen derived from one of said pathogens of interest;

(b) obtaining a fluorescently labelled immunoglobulin-reactive reagent;
(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample and the fluorescently labelled immunoglobulin-reactive reagent under conditions that will allow binding reactions between the antigens, the antibodies, and the fluorescently labelled immunoglobulin-reactive reagent wherein a reaction between a bead-bound antigen, an antibody in said fluid sample and a fluorescently labelled reagent causes an increase in a value of a fluorescent signal (F m) emitted from said bead;
(d) analyzing the reacted bead sample by said flow cytometry to determine the profile and the F m value of each bead analyzed;
(e) identifying the subset to which each bead belongs and therefore the antigen on said bead as a function of the profile of the classification parameter values;

and (f) detecting the antibodies in the fluid sample as a function of the identification in step (e) and the F m values.
43. The method of claim 42 wherein said antigens comprise one or more of the following antigens: Toxoplasma gondii, Rubella virus, Cytomegalovirus, and Herpes Simplex virus.
44. The method of claim 43 wherein said fluorescently labelled immunoglobulin-reactive reagent is anti-Human IgG.
45. The method of claim 43 wherein said fluorescently labelled immunoglobulin-reactive reagent is anti-Human IgM.
46. A method of flow cytometric assay for antibodies reactive with allergens of interest in a fluid sample comprising the steps of:
(a) obtaining a headset comprising a plurality of subsets of beads wherein the bead in each subset;
(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3 ... C n) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that a profile of the classification parameter values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameter values does not include a number of the beads in said each subset; and (2) are coupled to an antigen derived from an allergen of interest;
(b) obtaining a fluorescently labelled IgE reactive agent;
(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample and the fluorescently labelled IgE reactive agent under conditions that will allow binding reactions between the bead-bound antigens, the antibodies, and the fluorescently labelled IgE reactive agent wherein a reaction between a bead-bound antigen, an antibody in said fluid sample, and the fluorescently labelled IgE-reactive agent causes an increase in a value of a fluorescent signal (F m) emitted from said bead;
(d) analyzing the reacted bead sample by said flow cytometry to determine the profile and the F m value of each bead analyzed;
(e) identifying the subset to which each bead belongs and therefore the antigen on said bead as a function of the profile of the classification parameter values;

and (f) detecting the antibodies in the fluid sample as a function of the identification in step (e) and the F m values.
47. The method of claim 46 wherein said antigens comprise one or more of the following antigens: Junegrass, Red Top, Brome, Orchard, Timothy, Rye, Fesque, What, Quack, Bermuda, Johnson, Canary, Velvet, Saltgrass, Bahia, and Vernal.
48. The method of claim 46 wherein said fluorescently labelled IgE reactive agent is anti-human IgE.
49. The method of claim 46 wherein said fluorescently labelled IgE reactive agent is anti-canine IgE.
50. A method of flow cytometric analysis capable of quantitating a concentration of an analyte of interest in a fluid sample comprising:
(a) obtaining a beadset comprising subsets of beads, wherein the beads in each subset;
(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3 ... C n) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that a profile of the classification parameter values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameter values does not include a number of the beads in said each subset; and (2) are coupled to a reactant that will specifically react with the analyte of interest; and wherein the beads in a plurality of said subsets are coupled to the same reactant but at concentrations which differ among said subsets;
(b) mixing, to produce a reacted bead sample, the beadset with the fluid sample under conditions that will allow reactions between the analyte of interest and the reactants, wherein the reactions cause a change in a value of a fluorescent signal (F m) emitted from said bead;

(c) analyzing the reacted bead sample by said flow cytometry to determine the profile and the F m value of each bead analyzed;
(d) identifying the subset to which each bead belongs and therefore the concentration of the reactant coupled to the bead as a function of the profile of the classification parameter values; and (e) detecting the concentration of the analyte of interest in said fluid sample as a function of the identification in step (d) and the F m values of the beads in each of said subsets relative to F m values of a second set of the beads in each of said subsets, wherein said beads in said second set have not been reacted with said fluid sample but have been reacted with a known concentration of the analyte of interest.
51. A method of flow cytometric analysis capable of quantitating a concentration of an analyte of interest in a fluid sample comprising:
(a) obtaining a beadset comprising subsets of beads wherein the beads in each subset;
(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3 ... C n) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that a profile of the classification parameter values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameter values does not include a number of the beads in said each subset; and (2) are coupled to a reactant that will specifically react with the analyte of interest; and wherein the beads in a plurality of said subsets are coupled to the same reactant but at concentrations which differ among said subsets;
(b) mixing, to produce a reacted bead sample, the beadset, a fluorescently labelled competitive inhibitor of the reaction between the analyte of interest and the reactant on the beads, and the fluid sample under conditions that will allow reactions between the analyte of interest and the reactants on the beads in said set, wherein a reaction between an analyte of interest and a reactant on a bead causes a decrease in a value of a fluorescent signal (F m) emitted from said bead;

(c) analyzing the reacted sample by said flow cytometry to determine the profile and the F m value of each bead analyzed;

(d) identifying the subset to which each bead belongs and therefore the concentration of the reactant coupled to the bead as a function of the profile of the classification parameter values;

(e) assigning a bead subset value to each bead subset that correlates relatively with the concentration of analyte with which the bead subset was coupled;
(f) determining an inter-bead subset slope from a plot of mean F m for each bead subset versus bead subset value to produce an inter-bead subset slope; and (g) determining the concentration of the analyte of interest in the fluid sample by interpolation of the slope determined in step (f) into a standard assay curve wherein the inter-bead subset slopes of beads incubated with known concentrations of the analyte of interest are plotted against the log of the known concentration of the analyte of interest.
52. A method of generating a multiplexed standard assay curve for use in quantitating a concentration of an analyte of interest in a fluid sample comprising the steps of:
(a) obtaining a beadset comprising subsets of beads wherein the beads in each subset;
(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3 ... C n) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that a profile of the classification parameter values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameter values does not include a number of the beads in said each subset;

(2) are coupled to a reactant that will specifically react with a selected analyte of interest in the fluid sample; and (3) wherein the beads in a plurality of said subsets are coupled to the same reactant but at concentrations which differ among said subsets;
(b) mixing, to produce a reacted bead sample, the beadset, a fluorescently labelled competitive inhibitor of the analyte of interest, and a known concentration of the analyte of interest under conditions that will allow reactions between the analyte of interest and the reactants on the beads in said beadset, wherein a reaction between a reactant on a bead and an analyte of interest causes a decrease in a value of a fluorescent signal (F m) emitted from said bead;
(c) analyzing the reacted bead sample by said flow cytometry to determine the profile and the F m value of each bead analyzed;
(d) identifying the subset to which each bead belongs and therefore the concentration of the reactant with which the bead was coupled as a function of the profile of the classification parameter values; and (e) assigning a bead subset value to each bead subset that correlates relatively with the concentration of analyte coupled to the bead subset; and (f) determining an inter-bead subset slope from a plot of mean F m for each bead subset versus bead subset value; and (g) repeating steps (a) - (f) at least one time but with a known concentration of analyte of interest that differs from said concentration of analyte of interest employed in any other step (b); and (h) plotting to produce a standard curve the inter-bead subset slopes at each known concentration of analyte of interest against the log of each known concentration of analyte of interest.
53. A method for flow cytometric analysis to detect a plurality of nucleic acid analytes of interest in a single sample comprising:
(a) obtaining a beadset comprising a plurality of subsets of beads wherein the beads in each subset;

(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3 ... C n) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that a profile of the classification parameter values within each subset detectable by flow cytometry is unique, wherein the at least one of said classification parameter values does not include a number of the beads in said each subset;
(2) are coupled to a nucleic acid that will specifically hybridize with a selected nucleic acid analyte of interest in the single sample; and (3) wherein the nucleic acid coupled to the beads are reactive with a fluorescently labelled nucleic acid probe which competes with said selected nucleic acid analyte of interest for hybridization with said nucleic acids coupled to the beads;
(b) mixing, to produce a reacted bead sample, the beadset with the single sample under conditions that will allow hybridization between the nucleic acid analytes of interest and the nucleic acids coupled to the beads and thereby to allow the nucleic acid analytes of interest to inhibit hybridization between the fluorescently labelled nucleic acid probes with the nucleic acids coupled to said beads, resulting in a decrease in a fluorescent signal F m emitted from a bead with which a nucleic acid analyte of interest has reacted;
(c) analyzing the reacted bead sample by flow cytometry to determine the profile and an F m value of each bead analyzed;
(d) identifying the subset to which each bead belongs and therefore the nucleic acid on the bead as a function of the profile of the classification parameter values; and (e) detecting the presence or absence of the nucleic acid analytes of interest in said single sample as a function of the identification in step (d) and the F m values.
54. The method of claim 3 wherein said analytes of interest are enzymes, said reactants are fluorescent molecules which upon reaction with the enzymes lose fluorescence, and the F m values result from alteration of said fluorescent molecules.
55. The method of claim 3 wherein said analytes of interest are enzymes, said reactants are non-fluorescent molecules which upon reaction with the enzymes become fluorescent, and the F m values result from alteration of said non-fluorescent molecules.
56. The method of claim 3 wherein said analytes of interest are convertases which produce active anzymes from inactive precursors, said reactants are inactive precursors that are converted to active enzyme which in turn are reactants of fluorescently labelled substrates for said newly activated enzymes, and the F m values result from cleavage of said substrates from said beads.
57. The method of claim 5 wherein said analytes of interest are enzymes, said reactants are molecules which, upon reaction with the enzymes, become ligates for a fluorescently labelled ligand, and wherein the F m values result from reaction of the ligates with the fluorescently labelled ligand.
58. The method of claim 3 wherein said analyte is a cofactor which produces an active enzyme from an inactive apo-enzyme, said reactant is a fluorescently labelled substrate for said activated enzyme, and the F m values result from cleavage of said substrate from said active enzyme.
59. The method of claim 5 wherein the analytes of interest are DNA segments, the reactants on the beads are DNA segments capable of specifically hybridizing to said analytes, and the fluorescently labelled compounds are a fluorescent DNA segment also capable of specifically hybridizing with said reactant to compete with the hybridization of said reactant to said analytes of interest.
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US08/542,401 US5736330A (en) 1995-10-11 1995-10-11 Method and compositions for flow cytometric determination of DNA sequences
US08/542,401 1995-10-11
US08/540,814 1995-10-11
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Families Citing this family (326)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE43097E1 (en) 1994-10-13 2012-01-10 Illumina, Inc. Massively parallel signature sequencing by ligation of encoded adaptors
US6406848B1 (en) 1997-05-23 2002-06-18 Lynx Therapeutics, Inc. Planar arrays of microparticle-bound polynucleotides
US6251691B1 (en) 1996-04-25 2001-06-26 Bioarray Solutions, Llc Light-controlled electrokinetic assembly of particles near surfaces
US7041510B2 (en) 1996-04-25 2006-05-09 Bioarray Solutions Ltd. System and method for programmable illumination pattern generation
SE9602545L (en) 1996-06-25 1997-12-26 Michael Mecklenburg Method of discriminating complex biological samples
US20030027126A1 (en) 1997-03-14 2003-02-06 Walt David R. Methods for detecting target analytes and enzymatic reactions
US6327410B1 (en) 1997-03-14 2001-12-04 The Trustees Of Tufts College Target analyte sensors utilizing Microspheres
US6023540A (en) * 1997-03-14 2000-02-08 Trustees Of Tufts College Fiber optic sensor with encoded microspheres
US7622294B2 (en) 1997-03-14 2009-11-24 Trustees Of Tufts College Methods for detecting target analytes and enzymatic reactions
US6406845B1 (en) 1997-05-05 2002-06-18 Trustees Of Tuft College Fiber optic biosensor for selectively detecting oligonucleotide species in a mixed fluid sample
DE69838067T2 (en) 1997-05-23 2008-03-13 Bioarray Solutions Ltd. COLOR CODING AND SITU INQUIRY OF MATRIX-COUPLED CHEMICAL COMPOUNDS
US5948627A (en) 1997-05-30 1999-09-07 One Lambda Immunobead flow cytometric detection of anti-HLA panel-reactive antibody
US7115884B1 (en) 1997-10-06 2006-10-03 Trustees Of Tufts College Self-encoding fiber optic sensor
US7348181B2 (en) 1997-10-06 2008-03-25 Trustees Of Tufts College Self-encoding sensor with microspheres
EP1032701A1 (en) * 1997-10-28 2000-09-06 The Regents of The University of California Dna base mismatch detection using flow cytometry
US7271009B1 (en) 1997-11-18 2007-09-18 Bio-Rad Laboratories, Inc. Multi-analyte diagnostic test for thyroid disorders
AU1203199A (en) 1997-11-18 1999-06-07 Bio-Rad Laboratories, Inc. Multiplex flow immunoassays with magnetic particles as solid phase
CA2313047A1 (en) * 1997-12-04 1999-12-16 Nicholas Thomas Multiple assay method
US6210910B1 (en) 1998-03-02 2001-04-03 Trustees Of Tufts College Optical fiber biosensor array comprising cell populations confined to microcavities
US6455263B2 (en) 1998-03-24 2002-09-24 Rigel Pharmaceuticals, Inc. Small molecule library screening using FACS
AU3555599A (en) * 1998-04-13 1999-11-01 Luminex Corporation Liquid labeling with fluorescent microparticles
AU4700699A (en) * 1998-06-24 2000-01-10 Glaxo Group Limited Nucleotide detection method
EP1090293B2 (en) 1998-06-24 2019-01-23 Illumina, Inc. Decoding of array sensors with microspheres
US6642062B2 (en) 1998-09-03 2003-11-04 Trellis Bioinformatics, Inc. Multihued labels
EP1110090B1 (en) * 1998-09-03 2009-03-25 Trellis Bioscience, Inc. Multihued labels
US6429027B1 (en) 1998-12-28 2002-08-06 Illumina, Inc. Composite arrays utilizing microspheres
US6846460B1 (en) 1999-01-29 2005-01-25 Illumina, Inc. Apparatus and method for separation of liquid phases of different density and for fluorous phase organic syntheses
US6696304B1 (en) 1999-02-24 2004-02-24 Luminex Corporation Particulate solid phase immobilized protein quantitation
GB9905807D0 (en) * 1999-03-12 1999-05-05 Amersham Pharm Biotech Uk Ltd Analysis of differential gene expression
DK1923471T3 (en) * 1999-04-20 2013-04-02 Illumina Inc Detection of nucleic acid reactions on bead arrays
US6355431B1 (en) 1999-04-20 2002-03-12 Illumina, Inc. Detection of nucleic acid amplification reactions using bead arrays
US20060275782A1 (en) * 1999-04-20 2006-12-07 Illumina, Inc. Detection of nucleic acid reactions on bead arrays
AU4710800A (en) * 1999-05-12 2000-11-21 Axys Pharmaceuticals, Inc. Methods of software driven flow sorting for reiterative synthesis cycles
US6620584B1 (en) 1999-05-20 2003-09-16 Illumina Combinatorial decoding of random nucleic acid arrays
US6544732B1 (en) 1999-05-20 2003-04-08 Illumina, Inc. Encoding and decoding of array sensors utilizing nanocrystals
US8481268B2 (en) 1999-05-21 2013-07-09 Illumina, Inc. Use of microfluidic systems in the detection of target analytes using microsphere arrays
US8080380B2 (en) 1999-05-21 2011-12-20 Illumina, Inc. Use of microfluidic systems in the detection of target analytes using microsphere arrays
US6673554B1 (en) 1999-06-14 2004-01-06 Trellie Bioinformatics, Inc. Protein localization assays for toxicity and antidotes thereto
WO2001000875A2 (en) * 1999-06-25 2001-01-04 Motorola, Inc. Novel methods and products for arrayed microsphere analysis
ATE412184T1 (en) * 1999-08-17 2008-11-15 Luminex Corp METHOD FOR ANALYZING A PLURALITY OF SAMPLES OF DIFFERENT ORIGINS FOR ONE ANALYTE
EP1218545B1 (en) 1999-08-18 2012-01-25 Illumina, Inc. Methods for preparing oligonucleotide solutions
EP1212599A2 (en) 1999-08-30 2002-06-12 Illumina, Inc. Methods for improving signal detection from an array
WO2001020533A2 (en) * 1999-09-15 2001-03-22 Luminex Corporation Creation of a database of biochemical data and methods of use
WO2001021840A2 (en) * 1999-09-23 2001-03-29 Gene Logic, Inc. Indexing populations
JP3781934B2 (en) 1999-12-22 2006-06-07 株式会社ニチレイバイオサイエンス Enzyme-protein complex
AU3436601A (en) * 1999-12-23 2001-07-03 Illumina, Inc. Decoding of array sensors with microspheres
US7582420B2 (en) 2001-07-12 2009-09-01 Illumina, Inc. Multiplex nucleic acid reactions
US6913884B2 (en) 2001-08-16 2005-07-05 Illumina, Inc. Compositions and methods for repetitive use of genomic DNA
CA2399733C (en) 2000-02-07 2011-09-20 Illumina, Inc. Nucleic acid detection methods using universal priming
US7361488B2 (en) 2000-02-07 2008-04-22 Illumina, Inc. Nucleic acid detection methods using universal priming
WO2001057268A2 (en) 2000-02-07 2001-08-09 Illumina, Inc. Nucleic acid detection methods using universal priming
US6770441B2 (en) 2000-02-10 2004-08-03 Illumina, Inc. Array compositions and methods of making same
ATE412774T1 (en) 2000-02-16 2008-11-15 Illumina Inc PARALLEL GENOTYPING OF MULTIPLE PATIENT SAMPLES
JP2004500569A (en) * 2000-02-25 2004-01-08 ルミネックス コーポレイション Internal standards and controls for multiplex assays
WO2001073443A2 (en) * 2000-03-28 2001-10-04 The Government Of The United State Of America, As Represented By The Secretary Of The Department Of Health And Human Services Methods and compositions for the simultaneous detection of multiple analytes
EP1305632B1 (en) 2000-05-04 2011-07-27 Siemens Healthcare Diagnostics Products GmbH Methods for detection of multiple analytes
DE10065632A1 (en) 2000-05-12 2001-11-15 Smtech Biovision Holding Ag Ec Detecting polynucleotides by hybridizing an array of sequences on a carrier with detection probes, but not immobilized
DE50113940D1 (en) * 2000-05-12 2008-06-19 Gnothis Holding Sa PROCESS FOR DETECTION OF POLYNUCLEOTIDES
US9709559B2 (en) 2000-06-21 2017-07-18 Bioarray Solutions, Ltd. Multianalyte molecular analysis using application-specific random particle arrays
US7465540B2 (en) * 2000-09-21 2008-12-16 Luminex Corporation Multiple reporter read-out for bioassays
US20030045005A1 (en) 2000-10-17 2003-03-06 Michael Seul Light-controlled electrokinetic assembly of particles near surfaces
DE60219429T2 (en) * 2001-02-13 2008-01-03 Pronostics Ltd., Babraham BIOCHEMICAL PROCESS AND DEVICE FOR DETERMINING PROPERTIES OF PROTEINS
JP2004520052A (en) * 2001-02-13 2004-07-08 スマートビード テクノロジーズ リミティド Biochemical methods and devices for detecting genetic characteristics
WO2002084249A2 (en) 2001-04-10 2002-10-24 The Board Of Trustees Of The Leland Stanford Junior University Therapeutic and diagnostic uses of antibody specificity profiles
US7262063B2 (en) 2001-06-21 2007-08-28 Bio Array Solutions, Ltd. Directed assembly of functional heterostructures
US7473767B2 (en) 2001-07-03 2009-01-06 The Institute For Systems Biology Methods for detection and quantification of analytes in complex mixtures
US7479630B2 (en) 2004-03-25 2009-01-20 Bandura Dmitry R Method and apparatus for flow cytometry linked with elemental analysis
US8148171B2 (en) 2001-10-09 2012-04-03 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and methods
WO2003034029A2 (en) 2001-10-15 2003-04-24 Bioarray Solutions, Ltd. Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection
US6838289B2 (en) * 2001-11-14 2005-01-04 Beckman Coulter, Inc. Analyte detection system
EP2196544A1 (en) 2001-11-21 2010-06-16 Applied Biosystems, LLC Kit for ligation detection assays using codeable labels
US7232691B2 (en) * 2001-11-27 2007-06-19 Los Alamos National Security, Llc Bioassay and biomolecular identification, sorting, and collection methods using magnetic microspheres
US20030109067A1 (en) * 2001-12-06 2003-06-12 Immunetech, Inc. Homogeneous immunoassays for multiple allergens
US20030119073A1 (en) * 2001-12-21 2003-06-26 Stephen Quirk Sensors and methods of detection for proteinase enzymes
EP1900827A3 (en) 2002-05-21 2008-04-16 Bayer HealthCare AG Methods and compositions for the prediction, diagnosis, prognosis, prevention and treatment of malignant neoplasia
US7785898B2 (en) 2002-05-31 2010-08-31 Genetic Technologies Limited Maternal antibodies as fetal cell markers to identify and enrich fetal cells from maternal blood
US7923260B2 (en) 2002-08-20 2011-04-12 Illumina, Inc. Method of reading encoded particles
US7508608B2 (en) * 2004-11-17 2009-03-24 Illumina, Inc. Lithographically fabricated holographic optical identification element
US7872804B2 (en) * 2002-08-20 2011-01-18 Illumina, Inc. Encoded particle having a grating with variations in the refractive index
US7619819B2 (en) * 2002-08-20 2009-11-17 Illumina, Inc. Method and apparatus for drug product tracking using encoded optical identification elements
WO2004019277A1 (en) * 2002-08-20 2004-03-04 Cyvera Corporation Diffraction grating-based encoded micro-particles for multiplexed experiments
US7441703B2 (en) * 2002-08-20 2008-10-28 Illumina, Inc. Optical reader for diffraction grating-based encoded optical identification elements
US7164533B2 (en) * 2003-01-22 2007-01-16 Cyvera Corporation Hybrid random bead/chip based microarray
US7901630B2 (en) * 2002-08-20 2011-03-08 Illumina, Inc. Diffraction grating-based encoded microparticle assay stick
US7126755B2 (en) * 2002-09-12 2006-10-24 Moon John A Method and apparatus for labeling using diffraction grating-based encoded optical identification elements
US7900836B2 (en) * 2002-08-20 2011-03-08 Illumina, Inc. Optical reader system for substrates having an optically readable code
US7399643B2 (en) * 2002-09-12 2008-07-15 Cyvera Corporation Method and apparatus for aligning microbeads in order to interrogate the same
US20050227252A1 (en) * 2002-08-20 2005-10-13 Moon John A Diffraction grating-based encoded articles for multiplexed experiments
JP2005536769A (en) * 2002-08-20 2005-12-02 シヴェラ コーポレイション Optical identification elements based on diffraction gratings
AU2002950953A0 (en) * 2002-08-23 2002-09-12 Genera Biosystems Pty Ltd Coded nucleic acid carriers
CA2498913A1 (en) * 2002-09-12 2004-03-25 Cyvera Corporation Assay stick comprising coded microbeads
WO2004025561A1 (en) * 2002-09-12 2004-03-25 Cyvera Corporation Chemical synthesis using diffraction grating-based encoded optical elements
EP1540591A1 (en) * 2002-09-12 2005-06-15 Cyvera Corporation Diffraction grating-based encoded micro-particles for multiplexed experiments
US20100255603A9 (en) * 2002-09-12 2010-10-07 Putnam Martin A Method and apparatus for aligning microbeads in order to interrogate the same
US7092160B2 (en) * 2002-09-12 2006-08-15 Illumina, Inc. Method of manufacturing of diffraction grating-based optical identification element
US7526114B2 (en) 2002-11-15 2009-04-28 Bioarray Solutions Ltd. Analysis, secure access to, and transmission of array images
CA2513985C (en) 2003-01-21 2012-05-29 Illumina Inc. Chemical reaction monitor
ITMI20030339A1 (en) * 2003-02-26 2004-08-27 Pranha 50 Ltda METHOD, APPARATUS AND DEVICES FOR THE RENOVATION OF PIPES BY INTRODUCTION OF PLASTIC PIPES.
EP1599608A4 (en) 2003-03-05 2007-07-18 Genetic Technologies Ltd Identification of fetal dna and fetal cell markers in maternal plasma or serum
US8173443B2 (en) * 2003-03-31 2012-05-08 Women's And Children's Hospital Multiplex screening for lysosomal storage disorders (LSDs)
FR2855613B1 (en) * 2003-05-26 2005-08-19 Biocytex METHOD FOR DETECTION AND MULTIPLEX QUANTIFICATION OF ANALYTES IN A SAMPLE USING MICROSPHERES
US20040259100A1 (en) * 2003-06-20 2004-12-23 Illumina, Inc. Methods and compositions for whole genome amplification and genotyping
CA2528577C (en) 2003-06-20 2012-08-07 Illumina, Inc. Methods and compositions for whole genome amplification and genotyping
US20050181394A1 (en) * 2003-06-20 2005-08-18 Illumina, Inc. Methods and compositions for whole genome amplification and genotyping
AU2003903417A0 (en) 2003-07-04 2003-07-17 Genera Biosystems Pty Ltd Multiplex detection
US7260485B2 (en) * 2003-07-18 2007-08-21 Luminex Corporation Method and systems for distinguishing between materials having similar spectra
US8012768B2 (en) * 2003-07-18 2011-09-06 Bio-Rad Laboratories, Inc. System and method for multi-analyte detection
EP1656543B1 (en) 2003-07-18 2012-02-01 Bio-Rad Laboratories, Inc. System and method for multi-analyte detection
CN1860242B (en) * 2003-08-01 2013-11-06 戴诺生物技术有限公司 Self-hybridizing multiple target nucleic acid probes and methods of use
JP4491417B2 (en) * 2003-08-04 2010-06-30 エモリー・ユニバーシティ Porous material embedded with nano chemical species, method for producing the same, and method for using the same
US7692773B2 (en) * 2003-08-05 2010-04-06 Luminex Corporation Light emitting diode based measurement systems
US7069191B1 (en) 2003-08-06 2006-06-27 Luminex Corporation Methods for reducing the susceptibility of a peak search to signal noise
KR101166180B1 (en) * 2003-08-13 2012-07-18 루미넥스 코포레이션 Methods for controlling one or more parameters of a flow cytometer type measurement system
US20060057729A1 (en) * 2003-09-12 2006-03-16 Illumina, Inc. Diffraction grating-based encoded element having a substance disposed thereon
GB0321508D0 (en) * 2003-09-13 2003-10-15 Secr Defence Antibody-based identification of bacterial phenotypes
JP4639370B2 (en) * 2003-09-17 2011-02-23 ミリポア・コーポレイション Compositions and methods for analyzing a target analyte
TW200521436A (en) 2003-09-22 2005-07-01 Bioarray Solutions Ltd Surface immobilized polyelectrolyte with multiple functional groups capable of covalently bonding to biomolecules
EP1522594A3 (en) 2003-10-06 2005-06-22 Bayer HealthCare AG Methods and kits for investigating cancer
WO2005042763A2 (en) 2003-10-28 2005-05-12 Bioarray Solutions Ltd. Optimization of gene expression analysis using immobilized capture probes
US20050136414A1 (en) * 2003-12-23 2005-06-23 Kevin Gunderson Methods and compositions for making locus-specific arrays
EP1702216B1 (en) 2004-01-09 2015-11-04 Life Technologies Corporation Phosphor particle coded beads
KR20060123539A (en) 2004-01-14 2006-12-01 루미넥스 코포레이션 Methods for altering one or more parameters of a measurement system
CN101187621B (en) * 2004-01-14 2012-02-15 卢米尼克斯股份有限公司 Method and systems for dynamic range expansion
US7433123B2 (en) 2004-02-19 2008-10-07 Illumina, Inc. Optical identification element having non-waveguide photosensitive substrate with diffraction grating therein
WO2005082110A2 (en) * 2004-02-26 2005-09-09 Illumina Inc. Haplotype markers for diagnosing susceptibility to immunological conditions
US7229778B2 (en) 2004-02-26 2007-06-12 The Procter & Gamble Company Methods for determining the relative benefits and/or evaluating quantitative changes of products on epithelial tissue
EP1735619A4 (en) * 2004-04-01 2007-09-26 Rules Based Medicine Inc Universal shotgun assay
US7702466B1 (en) 2004-06-29 2010-04-20 Illumina, Inc. Systems and methods for selection of nucleic acid sequence probes
US7635563B2 (en) * 2004-06-30 2009-12-22 Massachusetts Institute Of Technology High throughput methods relating to microRNA expression analysis
US7848889B2 (en) 2004-08-02 2010-12-07 Bioarray Solutions, Ltd. Automated analysis of multiplexed probe-target interaction patterns: pattern matching and allele identification
EP1802710B1 (en) * 2004-10-12 2016-02-24 Luminex Corporation Methods for forming dyed microspheres and populations of dyed microspheres
US8088629B1 (en) 2004-10-12 2012-01-03 Luminex Corporation Methods for forming dyed microspheres and populations of microspheres
EP1802973B1 (en) * 2004-10-12 2010-05-05 Luminex Corporation Methods for altering surface characteristics of microspheres
EP1820001A1 (en) * 2004-11-12 2007-08-22 Luminex Corporation Methods and systems for positioning microspheres for imaging
US7604173B2 (en) * 2004-11-16 2009-10-20 Illumina, Inc. Holographically encoded elements for microarray and other tagging labeling applications, and method and apparatus for making and reading the same
DE602005019791D1 (en) 2004-11-16 2010-04-15 Illumina Inc METHOD AND DEVICE FOR READING CODED MICROBALLS
WO2006058334A2 (en) * 2004-11-29 2006-06-01 Perkinelmer Life And Analytical Sciences Prticle-based multiplex assay for identifying glycosylation
JP4974899B2 (en) * 2004-12-10 2012-07-11 ジェネラ バイオシステムズ リミテッド Composition and detection method
CA2591200A1 (en) * 2004-12-17 2006-06-22 Luminex Corporation Systems, illumination subsystems, and methods for increasing fluorescence emitted by a fluorophore
US20060160102A1 (en) 2005-01-18 2006-07-20 Hossein Fakhrai-Rad Identification of rare alleles by enzymatic enrichment of mismatched heteroduplexes
CA2775655C (en) * 2005-01-20 2014-03-25 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Multiplexed analysis for determining a serodiagnosis of viral infection
EP2244270B1 (en) 2005-01-20 2012-06-06 Luminex Corporation Microspheres having fluorescent and magnetic properties
EP1872128A4 (en) * 2005-04-20 2008-09-03 Becton Dickinson Co Multiplex microparticle system
JP4520361B2 (en) * 2005-05-25 2010-08-04 日立ソフトウエアエンジニアリング株式会社 Probe bead quality inspection method
DK2463386T3 (en) 2005-06-15 2017-07-31 Complete Genomics Inc Nucleic acid analysis using random mixtures of non-overlapping fragments
US20070043510A1 (en) * 2005-08-19 2007-02-22 Beckman Coulter, Inc. Assay system
US7504235B2 (en) 2005-08-31 2009-03-17 Kimberly-Clark Worldwide, Inc. Enzyme detection technique
US8031918B2 (en) 2005-09-21 2011-10-04 Luminex Corporation Methods and systems for image data processing
GB2432419A (en) 2005-11-16 2007-05-23 Agency Science Tech & Res Influenza A virus detection method
US7623624B2 (en) * 2005-11-22 2009-11-24 Illumina, Inc. Method and apparatus for labeling using optical identification elements characterized by X-ray diffraction
EP1969372A4 (en) * 2005-12-23 2009-04-22 Perkinelmer Las Inc Methods and compositions for detecting enzymatic activity
WO2007075891A2 (en) * 2005-12-23 2007-07-05 Perkinelmer Las, Inc. Multiplex assays using magnetic and non-magnetic particles
WO2007076128A2 (en) 2005-12-23 2007-07-05 Nanostring Technologies, Inc. Nanoreporters and methods of manufacturing and use thereof
WO2008018905A2 (en) * 2006-01-17 2008-02-14 Cellumen, Inc. Method for predicting biological systems responses
US8309025B1 (en) 2006-01-26 2012-11-13 Luminex Corporation Methods and systems for determining composition and completion of an experiment
WO2007093050A1 (en) 2006-02-13 2007-08-23 Olga Ornatsky Gene expression assays conducted by elemental analysis
CA2571904A1 (en) * 2006-02-15 2007-08-15 Fio Corporation System and method of detecting pathogens
US20070207513A1 (en) * 2006-03-03 2007-09-06 Luminex Corporation Methods, Products, and Kits for Identifying an Analyte in a Sample
US7914988B1 (en) 2006-03-31 2011-03-29 Illumina, Inc. Gene expression profiles to predict relapse of prostate cancer
US8758989B2 (en) 2006-04-06 2014-06-24 Kimberly-Clark Worldwide, Inc. Enzymatic detection techniques
US8124943B1 (en) 2006-04-06 2012-02-28 Lugade Ananda G Methods and systems for altering fluorescent intensities of a plurality of particles
US7830575B2 (en) * 2006-04-10 2010-11-09 Illumina, Inc. Optical scanner with improved scan time
JP2009533073A (en) * 2006-04-17 2009-09-17 ルミネックス・コーポレーション Methods, particles and kits for determining kinase activity
WO2007136724A2 (en) 2006-05-17 2007-11-29 Cellumen, Inc. Method for automated tissue analysis
US9810707B2 (en) * 2006-05-17 2017-11-07 Luminex Corporation Chip-based flow cytometer type systems for analyzing fluorescently tagged particles
CN101479603B (en) 2006-06-02 2012-11-21 卢米尼克斯股份有限公司 Systems and methods for performing measurements of one or more analytes comprising using magnetic particles and applying a magnetic field
US8296088B2 (en) 2006-06-02 2012-10-23 Luminex Corporation Systems and methods for performing measurements of one or more materials
EP2057465A4 (en) 2006-08-09 2010-04-21 Homestead Clinical Corp Organ-specific proteins and methods of their use
US8283624B2 (en) 2006-08-15 2012-10-09 Dvs Sciences Inc. Apparatus and method for elemental analysis of particles by mass spectrometry
US7501290B2 (en) * 2006-09-13 2009-03-10 Hanley Brian P Intraplexing method for improving precision of suspended microarray assays
CA2668410C (en) 2006-11-02 2017-05-16 Mitchell A. Winnik Particles containing detectable elemental code
WO2008061058A2 (en) 2006-11-10 2008-05-22 Luminex Corporation Flow cytometer and fluidic line assembly with multiple injection needles
US20100112602A1 (en) * 2006-11-10 2010-05-06 Taylor Lansing D Protein-Protein Interaction Biosensors and Methods of Use Thereof
SG143090A1 (en) * 2006-11-27 2008-06-27 Agency Science Tech & Res Influenza b virus detection method and kit therefor
AU2007333040B2 (en) 2006-12-13 2013-02-07 Luminex Corporation Systems and methods for multiplex analysis of PCR in real time
US7897360B2 (en) 2006-12-15 2011-03-01 Kimberly-Clark Worldwide, Inc. Enzyme detection techniques
CA2580589C (en) * 2006-12-19 2016-08-09 Fio Corporation Microfluidic detection system
US8003312B2 (en) * 2007-02-16 2011-08-23 The Board Of Trustees Of The Leland Stanford Junior University Multiplex cellular assays using detectable cell barcodes
US20080241831A1 (en) * 2007-03-28 2008-10-02 Jian-Bing Fan Methods for detecting small RNA species
WO2008119184A1 (en) 2007-04-02 2008-10-09 Fio Corporation System and method of deconvolving multiplexed fluorescence spectral signals generated by quantum dot optical coding technology
CN101821322B (en) 2007-06-22 2012-12-05 Fio公司 Systems and methods for manufacturing quantum dot-doped polymer microbeads
EP2395113A1 (en) 2007-06-29 2011-12-14 Population Genetics Technologies Ltd. Methods and compositions for isolating nucleic acid sequence variants
WO2009006739A1 (en) * 2007-07-09 2009-01-15 Fio Corporation Systems and methods for enhancing fluorescent detection of target molecules in a test sample
JP2010534322A (en) * 2007-07-23 2010-11-04 フィオ コーポレイション Methods and systems for collating, storing, analyzing, and accessing data collected and analyzed for biological and environmental analytes
US20090029347A1 (en) * 2007-07-27 2009-01-29 Thornthwaite Jerry T Method for Identifying Multiple Analytes Using Flow Cytometry
WO2009039165A1 (en) 2007-09-17 2009-03-26 Luminex Corporation Systems, storage mediums, and methods for identifying particles in flow
CN101861203B (en) 2007-10-12 2014-01-22 Fio公司 Flow focusing method and system for forming concentrated volumes of microbeads, and microbeads formed further thereto
WO2009086525A2 (en) * 2007-12-27 2009-07-09 Luminex Corporation Luminescent reporter modality for analyzing an assay
EP2240778B1 (en) * 2008-01-07 2014-07-23 Luminex Corporation Isolation and identification of cells from a complex sample matrix
CN101939648B (en) * 2008-01-07 2013-09-04 卢米耐克斯公司 Immunomagnetic capture and imaging of biological targets
CA2718276A1 (en) 2008-03-13 2009-09-17 Perkinelmer Las, Inc. Enzymatic substrates for multiple detection systems
JP2011519034A (en) * 2008-04-23 2011-06-30 ルミネックス コーポレーション How to create standards for multiple specimens found in starting materials of biological origin
WO2009155704A1 (en) 2008-06-25 2009-12-30 Fio Corporation Bio-threat alert system
CN104732199B (en) * 2008-07-17 2018-06-05 卢米耐克斯公司 For configuring the method for the specification area in classification matrix and storage medium
EP2331704B1 (en) 2008-08-14 2016-11-30 Nanostring Technologies, Inc Stable nanoreporters
CA2734321A1 (en) * 2008-08-15 2010-02-18 Litmus Rapid-B Llc Flow cytometry-based systems and methods for detecting microbes
MX2011002235A (en) 2008-08-29 2011-04-05 Fio Corp A single-use handheld diagnostic test device, and an associated system and method for testing biological and environmental test samples.
FR2935802B1 (en) * 2008-09-05 2012-12-28 Horiba Abx Sas METHOD AND DEVICE FOR CLASSIFYING, VISUALIZING AND EXPLORING BIOLOGICAL DATA
US8541207B2 (en) 2008-10-22 2013-09-24 Illumina, Inc. Preservation of information related to genomic DNA methylation
US8877511B2 (en) * 2008-11-10 2014-11-04 Luminex Corporation Method and system for manufacture and use of macroporous beads in a multiplex assay
CA2749660C (en) 2009-01-13 2017-10-31 Fio Corporation A handheld diagnostic test device and method for use with an electronic device and a test cartridge in a rapid diagnostic test
US20100220315A1 (en) * 2009-02-27 2010-09-02 Beckman Coulter, Inc. Stabilized Optical System for Flow Cytometry
US20110046919A1 (en) 2009-03-02 2011-02-24 Juliesta Elaine Sylvester Method for accurate measurement of enzyme activities
US20100261292A1 (en) * 2009-04-10 2010-10-14 Meso Scale Technologies, Llc Methods for Conducting Assays
EP2421955A4 (en) 2009-04-21 2012-10-10 Genetic Technologies Ltd Methods for obtaining fetal genetic material
US8343775B2 (en) * 2009-05-06 2013-01-01 Hanley Brian P Method and device for affinity differential intraplexing
EP2290102A1 (en) 2009-07-08 2011-03-02 Administración General De La Communidad Autónoma De Euskadi Methods for the diagnosis of multiple sclerosis based on its microRNA expression profiling
EP2488876B1 (en) 2009-10-13 2017-03-01 Nanostring Technologies, Inc Protein detection via nanoreporters
GB2475226A (en) 2009-11-03 2011-05-18 Genetic Analysis As Universal Prokaryote 16S ribosome PCR primer pair
EP2336769A1 (en) 2009-12-18 2011-06-22 F. Hoffmann-La Roche AG Trigger assay for differentiating between rheumatic and non-rheumatic disorders
CA2785529C (en) 2010-02-11 2019-01-08 Nanostring Technologies, Inc. Compositions and methods for the detection of small rnas
GB201002627D0 (en) 2010-02-16 2010-03-31 Loxbridge Res Llp Aptamer based analyte detection method
AU2011221243B2 (en) 2010-02-25 2016-06-02 Advanced Liquid Logic, Inc. Method of making nucleic acid libraries
US8747677B2 (en) 2010-04-09 2014-06-10 Luminex Corporation Magnetic separation device
EP2558596B1 (en) 2010-04-16 2018-03-14 The Government Of The United States Of America As Reresented By The Secretary Of The Department Of Health & Human Services Real time pcr assay for detection of bacterial respiratory pathogens
US9228240B2 (en) 2010-06-03 2016-01-05 California Institute Of Technology Methods for detecting and quantifying viable bacterial endo-spores
US8274656B2 (en) 2010-06-30 2012-09-25 Luminex Corporation Apparatus, system, and method for increasing measurement accuracy in a particle imaging device
US8767069B2 (en) 2010-06-30 2014-07-01 Luminex Corporation Apparatus, system, and method for increasing measurement accuracy in a particle imaging device using light distribution
EP2628004A4 (en) 2010-10-14 2014-08-20 Meso Scale Technologies Llc Reagent storage in an assay device
EP2633082B1 (en) 2010-10-26 2018-08-15 The Government of the United States of America as Represented by the Secretary of the Department of Health and Human Services Rapid salmonella serotyping assay
US9115408B2 (en) 2010-10-26 2015-08-25 The United States Of America As Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention Rapid Salmonella serotyping assay
WO2012068055A2 (en) 2010-11-17 2012-05-24 Advanced Liquid Logic, Inc. Capacitance detection in a droplet actuator
GB201021397D0 (en) 2010-12-16 2011-01-26 Genetic Analysis As Diagnosis of Crohn's disease
GB201021399D0 (en) 2010-12-16 2011-01-26 Genetic Analysis As Oligonucleotide probe set and methods of microbiota profiling
CA2860690A1 (en) * 2011-01-05 2012-07-12 Zeus Scientific, Inc. Diagnostic methods
GB201102693D0 (en) 2011-02-16 2011-03-30 Genetic Analysis As Method for identifying neonates at risk for necrotizing enterocolitis
WO2012125807A2 (en) 2011-03-17 2012-09-20 Cernostics, Inc. Systems and compositions for diagnosing barrett's esophagus and methods of using the same
WO2012135340A2 (en) 2011-03-28 2012-10-04 Nanostring Technologies, Inc. Compositions and methods for diagnosing cancer
CN103562729A (en) 2011-05-02 2014-02-05 先进流体逻辑公司 Molecular diagnostics platform
EP2707131B1 (en) 2011-05-09 2019-04-24 Advanced Liquid Logic, Inc. Microfluidic feedback using impedance detection
WO2012154794A2 (en) 2011-05-10 2012-11-15 Advanced Liquid Logic, Inc. Enzyme concentration and assays
EP2546358A1 (en) 2011-07-15 2013-01-16 Laboratorios Del. Dr. Esteve, S.A. Methods and reagents for efficient control of HIV progression
WO2013049613A1 (en) 2011-09-29 2013-04-04 Luminex Corporation Hydrolysis probes
KR101979287B1 (en) * 2011-10-18 2019-08-28 루미넥스 코포레이션 Methods and systems for image data processing
DK2776833T3 (en) 2011-11-11 2019-01-02 Eli N Glezer Cobinder-supported testing method
WO2013078216A1 (en) 2011-11-21 2013-05-30 Advanced Liquid Logic Inc Glucose-6-phosphate dehydrogenase assays
WO2013083847A2 (en) 2011-12-09 2013-06-13 Institut Pasteur Multiplex immuno screening assay
WO2013119763A1 (en) 2012-02-07 2013-08-15 Intuitive Biosciences, Inc. Mycobacterium tuberculosis specific peptides for detection of infection or immunization in non-human primates
US20130274125A1 (en) * 2012-04-16 2013-10-17 Bio-Rad Laboratories Inc. Multiplex immunoassay for rheumatoid arthritis and other autoimmune diseases
US9012022B2 (en) 2012-06-08 2015-04-21 Illumina, Inc. Polymer coatings
ES2434853B1 (en) 2012-06-12 2014-09-30 Fundación Centro Nacional De Investigaciones Cardiovasculares Carlos Iii Molecular marker of therapeutic potency of human mesenchymal stem cells and their uses
US9394574B2 (en) 2012-06-12 2016-07-19 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Methods for detecting Legionella nucleic acids in a sample
WO2013188857A2 (en) 2012-06-15 2013-12-19 Luminex Corporation Apparatus, system, and method for image normalization using a gaussian residual of fit selection criteria
AU2014249190C1 (en) 2013-03-11 2021-11-18 Meso Scale Technologies, Llc. Improved methods for conducting multiplexed assays
WO2014165061A1 (en) 2013-03-13 2014-10-09 Meso Scale Technologies, Llc. Improved assay methods
US10114015B2 (en) 2013-03-13 2018-10-30 Meso Scale Technologies, Llc. Assay methods
CN105358708A (en) 2013-03-14 2016-02-24 儿童医学中心公司 Use of cd36 to identify cancer subjects for treatment
JP6333297B2 (en) 2013-03-15 2018-05-30 イルミナ ケンブリッジ リミテッド Modified nucleoside or modified nucleotide
CA2942831A1 (en) 2013-03-15 2014-09-18 The Trustees Of Princeton University Methods and devices for high throughput purification
EP2971287B1 (en) 2013-03-15 2019-08-14 GPB Scientific, LLC On-chip microfluidic processing of particles
EP2970631B1 (en) 2013-03-15 2017-05-03 Lubrizol Advanced Materials, Inc. Heavy metal free cpvc compositions
US20150064153A1 (en) 2013-03-15 2015-03-05 The Trustees Of Princeton University High efficiency microfluidic purification of stem cells to improve transplants
US10502678B2 (en) * 2013-03-15 2019-12-10 Beckman Coulter, Inc. Systems and methods for panel design in flow cytometry
EP2787349A1 (en) 2013-04-03 2014-10-08 Affiris AG Method for detecting proteinopathy-specific antibodies in a biological sample
EP2787348A1 (en) * 2013-04-03 2014-10-08 Affiris AG Method for detecting aSyn-specific antibodies in a biological sample
EP2787347A1 (en) * 2013-04-03 2014-10-08 Affiris AG Method for detecting Aß-specific antibodies in a biological sample
CN103235120B (en) * 2013-04-22 2014-12-31 苏州华益美生物科技有限公司 Kit for compound detection of hepatitis E virus antibody profile as well as application of kit
ES2523016B1 (en) 2013-05-20 2015-09-09 3P Biopharmaceuticals Alpha-viral vectors and cell lines for the production of recombinant proteins
EP3011056B1 (en) 2013-06-19 2019-03-06 Luminex Corporation Real-time multiplexed hydrolysis probe assay
KR102266002B1 (en) 2013-07-01 2021-06-16 일루미나, 인코포레이티드 Catalyst-free surface functionalization and polymer grafting
EP3461913B1 (en) 2013-08-09 2020-06-24 Luminex Corporation Probes for improved melt discrimination and multiplexing in nucleic acid assays
US10124351B2 (en) 2013-08-13 2018-11-13 Advanced Liquid Logic, Inc. Methods of improving accuracy and precision of droplet metering using an on-actuator reservoir as the fluid input
EP3726213A1 (en) 2013-08-19 2020-10-21 Singular Bio Inc. Assays for single molecule detection and use thereof
EP3038834B1 (en) 2013-08-30 2018-12-12 Illumina, Inc. Manipulation of droplets on hydrophilic or variegated-hydrophilic surfaces
BR112016008975B1 (en) 2013-10-21 2023-02-28 Takeda Pharmaceutical Company Limited METHODS TO DETECT, MONITOR THE DEVELOPMENT AND EVALUATE THE EFFECTIVENESS OF A TREATMENT OF AN AUTOIMMUNE DISEASE
CN105874385B (en) 2013-12-19 2021-08-20 Illumina公司 Substrate comprising a nanopatterned surface and method for the production thereof
AU2015206336B2 (en) 2014-01-16 2020-01-23 Illumina, Inc. Gene expression panel for prognosis of prostate cancer recurrence
AU2015249592A1 (en) 2014-04-24 2016-12-15 Immusant, Inc. Use of Interleukin-2 for diagnosis of Celiac disease
AU2015253299B2 (en) 2014-04-29 2018-06-14 Illumina, Inc. Multiplexed single cell gene expression analysis using template switch and tagmentation
WO2015175856A1 (en) 2014-05-15 2015-11-19 Meso Scale Technologies, Llc. Improved assay methods
GB201414098D0 (en) 2014-08-08 2014-09-24 Illumina Cambridge Ltd Modified nucleotide linkers
KR102465287B1 (en) 2014-08-11 2022-11-09 루미넥스 코포레이션 Probes for improved melt discrimination and multiplexing in nucleic acid assays
EP3183577B1 (en) 2014-08-21 2020-08-19 Illumina Cambridge Limited Reversible surface functionalization
US20160097082A1 (en) 2014-09-23 2016-04-07 Ohmx Corporation Prostate specific antigen proteolytic activity for clinical use
WO2016061509A1 (en) 2014-10-17 2016-04-21 The Broad Institute, Inc. Compositions and methods of treatng muscular dystrophy
PT3212684T (en) 2014-10-31 2020-02-03 Illumina Cambridge Ltd Novel polymers and dna copolymer coatings
CN107531767A (en) 2014-11-21 2018-01-02 免疫桑特公司 For treating and diagnosing the peptide used in type 1 diabetes
US10576471B2 (en) 2015-03-20 2020-03-03 Illumina, Inc. Fluidics cartridge for use in the vertical or substantially vertical position
ES2945607T3 (en) 2015-07-17 2023-07-04 Illumina Inc Polymer sheets for sequencing applications
SG10202107055SA (en) 2015-07-17 2021-08-30 Nanostring Technologies Inc Simultaneous quantification of a plurality of proteins in a user-defined region of a cross-sectioned tissue
KR102608653B1 (en) 2015-07-17 2023-11-30 나노스트링 테크놀로지스, 인크. Simultaneous quantification of gene expression in user-defined regions of sectioned tissue
US10976232B2 (en) 2015-08-24 2021-04-13 Gpb Scientific, Inc. Methods and devices for multi-step cell purification and concentration
AU2016316773B2 (en) 2015-08-28 2020-01-30 Illumina, Inc. Nucleic acid sequence analysis from single cells
EP4152001A1 (en) 2015-11-25 2023-03-22 Cernostics, Inc. Methods of predicting progression of barrett's esophagus
ES2786974T3 (en) 2016-04-07 2020-10-14 Illumina Inc Methods and systems for the construction of standard nucleic acid libraries
EP3458913B1 (en) 2016-05-18 2020-12-23 Illumina, Inc. Self assembled patterning using patterned hydrophobic surfaces
US20170336397A1 (en) 2016-05-19 2017-11-23 Roche Molecular Systems, Inc. RFID Detection Systems And Methods
WO2018089764A1 (en) 2016-11-11 2018-05-17 Ascendant Dx, Llc Compositions and methods for diagnosing and differentiating systemic juvenile idiopathic arthritis and kawasaki disease
KR102476709B1 (en) 2016-11-21 2022-12-09 나노스트링 테크놀로지스, 인크. Chemical compositions and methods of using same
JP2020536115A (en) 2017-10-04 2020-12-10 オプコ ファーマシューティカルズ、エルエルシー Articles and methods for personalized cancer therapy
EP3490237A1 (en) 2017-11-23 2019-05-29 Viramed Biotech AG Device and method for parallel evaluation of multiple test areas
ES2937927T3 (en) 2018-01-29 2023-04-03 St Jude Childrens Res Hospital Inc Method for nucleic acid amplification
CA3090699A1 (en) 2018-02-12 2019-08-15 Nanostring Technologies, Inc. Biomolecular probes and methods of detecting gene and protein expression
WO2019165304A1 (en) 2018-02-23 2019-08-29 Meso Scale Technologies, Llc. Methods of screening antigen-binding molecules by normalizing for the concentration of antigen-binding molecule
KR20210061962A (en) 2018-05-14 2021-05-28 나노스트링 테크놀로지스, 인크. Chemical composition and method of use thereof
WO2019222708A2 (en) 2018-05-17 2019-11-21 Meso Scale Technologies, Llc. Methods for isolating surface marker displaying agents
US10936163B2 (en) 2018-07-17 2021-03-02 Methodical Mind, Llc. Graphical user interface system
US11929171B2 (en) 2018-10-18 2024-03-12 The Board Of Trustees Of The Leland Stanford Junior University Methods for evaluation and treatment of glycemic dysregulation and atherosclerotic cardiovascular disease and applications thereof
US20210382043A1 (en) 2018-10-23 2021-12-09 Meso Scale Technologies, Llc. Methods for isolating surface marker displaying agents
US11293061B2 (en) 2018-12-26 2022-04-05 Illumina Cambridge Limited Sequencing methods using nucleotides with 3′ AOM blocking group
CA3132154A1 (en) 2019-03-01 2020-09-10 Meso Scale Technologies, Llc. Electrochemiluminescent labeled probes for use in immunoassay methods, methods using such and kits comprising same
US11421271B2 (en) 2019-03-28 2022-08-23 Illumina Cambridge Limited Methods and compositions for nucleic acid sequencing using photoswitchable labels
MX2022012184A (en) 2020-03-30 2022-10-27 Illumina Inc Methods and compositions for preparing nucleic acid libraries.
CN115867560A (en) 2020-06-22 2023-03-28 伊鲁米纳剑桥有限公司 Nucleosides and nucleotides having 3' acetal capping groups
EP4182687A2 (en) * 2020-07-20 2023-05-24 Bio-Rad Laboratories, Inc. Immunoassay for sars-cov-2 neutralizing antibodies and materials therefor
US20220033900A1 (en) 2020-07-28 2022-02-03 Illumina Cambridge Limited Substituted coumarin dyes and uses as fluorescent labels
US20230349920A1 (en) 2020-09-04 2023-11-02 Meso Scale Technologies, Llc. Methods for isolating central nervous system surface marker displaying agents
US20220195516A1 (en) 2020-12-17 2022-06-23 Illumina Cambridge Limited Methods, systems and compositions for nucleic acid sequencing
US20220195196A1 (en) 2020-12-17 2022-06-23 Illumina Cambridge Limited Alkylpyridinium coumarin dyes and uses in sequencing applications
US20220195517A1 (en) 2020-12-17 2022-06-23 Illumina Cambridge Limited Long stokes shift chromenoquinoline dyes and uses in sequencing applications
US20220195518A1 (en) 2020-12-22 2022-06-23 Illumina Cambridge Limited Methods and compositions for nucleic acid sequencing
WO2022150711A1 (en) 2021-01-11 2022-07-14 Meso Scale Technologies, Llc. Assay system calibration systems and methods
WO2022165167A1 (en) * 2021-01-29 2022-08-04 Flowmetric Life Sciences, Inc. Detecting sars-cov-2 and other infective agents
EP4330672A1 (en) 2021-04-26 2024-03-06 Meso Scale Technologies, LLC. Methods for isolating and analyzing a target analyte encapsulated by a surface marker displaying agent
EP4334327A1 (en) 2021-05-05 2024-03-13 Illumina Cambridge Limited Fluorescent dyes containing bis-boron fused heterocycles and uses in sequencing
CA3216735A1 (en) 2021-05-20 2022-11-24 Patrizia IAVICOLI Compositions and methods for sequencing by synthesis
WO2022265994A1 (en) 2021-06-15 2022-12-22 Illumina, Inc. Hydrogel-free surface functionalization for sequencing
WO2023004339A1 (en) 2021-07-21 2023-01-26 Methodical Mind, Llc. Graphical user interface system guide module
WO2023004357A1 (en) 2021-07-23 2023-01-26 Illumina, Inc. Methods for preparing substrate surface for dna sequencing
WO2023186815A1 (en) 2022-03-28 2023-10-05 Illumina Cambridge Limited Labeled avidin and methods for sequencing
AU2023246772A1 (en) 2022-03-29 2024-01-18 Illumina Inc. Chromenoquinoline dyes and uses in sequencing
WO2023212315A2 (en) 2022-04-29 2023-11-02 Meso Scale Technologies, Llc. Methods for detecting and isolating extracellular vesicles
WO2023232829A1 (en) 2022-05-31 2023-12-07 Illumina, Inc Compositions and methods for nucleic acid sequencing
US20230416279A1 (en) 2022-06-28 2023-12-28 Illumina Cambridge Limited Fluorescent dyes containing fused tetracyclic bis-boron heterocycle and uses in sequencing
WO2024039516A1 (en) 2022-08-19 2024-02-22 Illumina, Inc. Third dna base pair site-specific dna detection
CN116794313B (en) * 2023-08-18 2023-11-03 江西赛基生物技术有限公司 Kit and method for simultaneously detecting three tumor markers based on flow cytometry

Family Cites Families (88)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4166105A (en) 1973-07-30 1979-08-28 Block Engineering, Inc. Dye tagged reagent
GB1451669A (en) 1974-01-02 1976-10-06 Radiochemical Centre Ltd Protein staining
US4108972A (en) 1974-03-15 1978-08-22 Dreyer William J Immunological reagent employing radioactive and other tracers
US3959650A (en) 1974-08-26 1976-05-25 Intelcom Rad Tech Method for detecting and identifying allergy
SE7412164L (en) 1974-09-27 1976-03-29 Pharmacia Ab FUNDS FOR INTRAVASCULAR ADMINISTRATION
US3952091A (en) 1974-10-23 1976-04-20 Hoffmann-La Roche Inc. Simultaneous multiple radioimmunoassay
US4169137A (en) 1974-12-20 1979-09-25 Block Engineering, Inc. Antigen detecting reagents
US4075462A (en) * 1975-01-08 1978-02-21 William Guy Rowe Particle analyzer apparatus employing light-sensitive electronic detector array
US4010250A (en) 1975-03-06 1977-03-01 The United States Of America As Represented By The Secretary Of The Navy Radioactive iodine (125I) labeling of latex particles
US4166102A (en) 1975-04-07 1979-08-28 Becton, Dickinson And Company Immobilized immunoadsorbent
US4018884A (en) 1975-06-26 1977-04-19 Hoffmann-La Roche Inc. Fluorogenic materials and labeling techniques
US4341957A (en) 1975-11-26 1982-07-27 Analytical Radiation Corporation Fluorescent antibody composition for immunofluorometric assay
US4317810A (en) 1975-09-29 1982-03-02 Cordis Laboratories, Inc. Waffle-like matrix for immunoassay and preparation thereof
US4201763A (en) 1975-10-09 1980-05-06 Bio-Rad Laboratories, Inc. Solid phase immunofluorescent assay method
US4113433A (en) 1975-12-15 1978-09-12 Gyaneshwar Prasad Khare Radioimmunoassay of hormones and metabolites in blood serum and plasma
US4028056A (en) 1976-04-20 1977-06-07 Technicon Instruments Corporation Substance separation technique
GB1582956A (en) 1976-07-30 1981-01-21 Ici Ltd Composite magnetic particles
US4108974A (en) 1976-08-27 1978-08-22 Bio-Rad Laboratories, Inc. Radioimmunoassay for thyroid hormone
US4090850A (en) 1976-11-01 1978-05-23 E. R. Squibb & Sons, Inc. Apparatus for use in radioimmunoassays
US4088746A (en) 1976-11-11 1978-05-09 Bio-Rad Laboratories, Inc. Radioimmunoassay for thyroid-stimulating hormone (TSH)
US4182750A (en) 1977-04-21 1980-01-08 Sullivan Thomas E Bloodcompatible functional polymers
DE2720704C2 (en) 1977-05-07 1986-09-25 Behringwerke Ag, 3550 Marburg New glycoprotein, process for its production and its uses
US4115535A (en) 1977-06-22 1978-09-19 General Electric Company Diagnostic method employing a mixture of normally separable protein-coated particles
US4219335A (en) 1978-09-18 1980-08-26 E. I. Du Pont De Nemours And Company Immunochemical testing using tagged reagents
US4184849A (en) 1977-12-05 1980-01-22 Technicon Instruments Corporation Mixed agglutination
US4231750A (en) 1977-12-13 1980-11-04 Diagnostic Reagents, Inc. Methods for performing chemical assays using fluorescence and photon counting
US4283382A (en) 1977-12-28 1981-08-11 Eastman Kodak Company Fluorescent labels comprising rare earth chelates
US4225783A (en) 1978-07-24 1980-09-30 Abbott Laboratories Determination of microbial cells in aqueous samples
US4302536A (en) 1978-08-15 1981-11-24 Longenecker Robert W Colorimetric immunoassay process
US4244940A (en) 1978-09-05 1981-01-13 Bio-Rad Laboratories, Inc. Single-incubation two-site immunoassay
US4259313A (en) 1978-10-18 1981-03-31 Eastman Kodak Company Fluorescent labels
US4342739A (en) 1979-01-09 1982-08-03 Fuji Photo Film Co., Ltd. Novel material for immunological assay of biochemical components and a process for the determination of said components
US4278653A (en) 1979-02-01 1981-07-14 New England Nuclear Corporation Methods and kits for double antibody immunoassay providing a colored pellet for easy visualization
US4254096A (en) 1979-10-04 1981-03-03 Bio-Rad Laboratories, Inc. Reagent combination for solid phase immunofluorescent assay
US4340564A (en) 1980-07-21 1982-07-20 Daryl Laboratories, Inc. Immunoadsorptive surface coating for solid-phase immunosubstrate and solid-phase immunosubstrate
US4673288A (en) 1981-05-15 1987-06-16 Ratcom, Inc. Flow cytometry
US4499052A (en) * 1982-08-30 1985-02-12 Becton, Dickinson And Company Apparatus for distinguishing multiple subpopulations of cells
JPS59174742A (en) 1983-03-25 1984-10-03 Agency Of Ind Science & Technol Method and apparatus for dividing and sorting fine particle
US4713348A (en) 1983-04-05 1987-12-15 Syntex (U.S.A.) Inc. Fluorescent multiparameter particle analysis
DE3322373C2 (en) 1983-05-19 1986-12-04 Ioannis Dr. 3000 Hannover Tripatzis Test means and methods for the detection of antigens and / or antibodies
US4710021A (en) 1983-10-14 1987-12-01 Sequoia-Turner Corporation Particulate matter analyzing apparatus and method
US4665020A (en) 1984-05-30 1987-05-12 United States Department Of Energy Flow cytometer measurement of binding assays
US4661913A (en) 1984-09-11 1987-04-28 Becton, Dickinson And Company Apparatus and method for the detection and classification of articles using flow cytometry techniques
US4676640A (en) 1984-09-12 1987-06-30 Syntex (U.S.A.) Inc. Fluctuation analysis for enhanced particle detection
US4857451A (en) 1984-12-24 1989-08-15 Flow Cytometry Standards Corporation Method of compensating and calibrating a flow cytometer, and microbead standards kit therefor
US5093234A (en) 1984-12-24 1992-03-03 Caribbean Microparticles Corporation Method of aligning, compensating, and calibrating a flow cytometer for analysis of samples, and microbead standards kit therefor
US5380663A (en) * 1984-12-24 1995-01-10 Caribbean Microparticles Corporation Automated system for performance analysis and fluorescence quantitation of samples
US4918004A (en) 1984-12-24 1990-04-17 Caribbean Microparticles Corporation Method of calibrating a flow cytometer or fluorescence microscope for quantitating binding antibodies on a selected sample, and microbead calibration kit therefor
US4884886A (en) 1985-02-08 1989-12-05 The United States Of America As Represented By The Department Of Energy Biological particle identification apparatus
ES8706660A1 (en) 1985-04-23 1987-07-01 Hoechst Roussel Pharma (Aminoalkylthio)hydroxydibenzoxepins, a process for their preparation and their use as medicaments.
EP0200113A3 (en) * 1985-04-30 1987-03-18 Pandex Laboratories, Inc. A method of solid phase nucleic acid hybridization assay incorporating a luminescent label
US4868104A (en) 1985-09-06 1989-09-19 Syntex (U.S.A.) Inc. Homogeneous assay for specific polynucleotides
US5281517A (en) * 1985-11-04 1994-01-25 Cell Analysis Systems, Inc. Methods for immunoploidy analysis
US4767205A (en) 1986-01-28 1988-08-30 Flow Cytometry Standards Corporation Composition and method for hidden identification
CA1301606C (en) * 1986-05-02 1992-05-26 David H. Gillespie Chaotropic method for evaluating nucleic acids in a biological sample
US5319079A (en) 1986-05-15 1994-06-07 Beckman Instruments, Inc. Process for terminal substituting of a polynucleotide
SE458968B (en) * 1987-06-16 1989-05-22 Wallac Oy BIOSPECIFIC ANALYTICAL PROCEDURE FOR MULTIPLE ANALYTICS WHICH DO NOT INCLUDE PARTICULAR COATING AND LABELING WITH FLUORESCING LABEL SUBSTANCES
US4987539A (en) * 1987-08-05 1991-01-22 Stanford University Apparatus and method for multidimensional characterization of objects in real time
US5403711A (en) 1987-11-30 1995-04-04 University Of Iowa Research Foundation Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is cleaved
US4887721A (en) 1987-11-30 1989-12-19 The United States Of America As Represented By The United States Department Of Energy Laser particle sorter
US5104791A (en) * 1988-02-09 1992-04-14 E. I. Du Pont De Nemours And Company Particle counting nucleic acid hybridization assays
US5385822A (en) 1988-05-02 1995-01-31 Zynaxis, Inc. Methods for detection and quantification of cell subsets within subpopulations of a mixed cell population
NO164622C (en) * 1988-05-11 1990-10-24 Tore Lindmo BINAER IMMUNOMETRIC PARTICLE-BASED METHOD FOR MEASURING SPECIFIC SERUM ANTIGENS USING LIQUID FLOW MICROPHOTOMETRY AND A PREPARED TARGET SET UP THEREOF.
US4905169A (en) 1988-06-02 1990-02-27 The United States Of America As Represented By The United States Department Of Energy Method and apparatus for simultaneously measuring a plurality of spectral wavelengths present in electromagnetic radiation
US5149661A (en) 1988-06-08 1992-09-22 Sarasep, Inc. Fluid analysis with particulate reagent suspension
US5408307A (en) 1988-07-11 1995-04-18 Omron Tateisi Electronics Co. Cell analyzer
FR2638848B1 (en) 1988-11-04 1993-01-22 Chemunex Sa METHOD OF DETECTION AND / OR DETERMINATION IN A LIQUID OR SEMI-LIQUID MEDIUM OF AT LEAST ONE ORGANIC, BIOLOGICAL OR MEDICINAL SUBSTANCE, BY AN AGGLUTINATION METHOD
GB8902689D0 (en) * 1989-02-07 1989-03-30 Ici Plc Assay method
US5156810A (en) 1989-06-15 1992-10-20 Biocircuits Corporation Biosensors employing electrical, optical and mechanical signals
CA1341094C (en) 1989-09-25 2000-09-05 Ronald G. Worton Diagnosis for malignant hyperthermia
US5107422A (en) 1989-10-02 1992-04-21 Kamentsky Louis A Method and apparatus for measuring multiple optical properties of biological specimens
US5401847A (en) 1990-03-14 1995-03-28 Regents Of The University Of California DNA complexes with dyes designed for energy transfer as fluorescent markers
US5150313A (en) 1990-04-12 1992-09-22 Regents Of The University Of California Parallel pulse processing and data acquisition for high speed, low error flow cytometry
US5326692B1 (en) 1992-05-13 1996-04-30 Molecular Probes Inc Fluorescent microparticles with controllable enhanced stokes shift
US5224058A (en) 1990-05-01 1993-06-29 Becton, Dickinson And Company Method for data transformation
US5273881A (en) 1990-05-07 1993-12-28 Daikin Industries, Ltd. Diagnostic applications of double D-loop formation
US5127730A (en) 1990-08-10 1992-07-07 Regents Of The University Of Minnesota Multi-color laser scanning confocal imaging system
US5204884A (en) 1991-03-18 1993-04-20 University Of Rochester System for high-speed measurement and sorting of particles
WO1992017853A2 (en) * 1991-04-05 1992-10-15 Pattern Recognition, L.P. Direct data base analysis, forecasting and diagnosis method
US5199576A (en) 1991-04-05 1993-04-06 University Of Rochester System for flexibly sorting particles
US5286452A (en) 1991-05-20 1994-02-15 Sienna Biotech, Inc. Simultaneous multiple assays
EP0594763B1 (en) * 1991-07-16 1998-09-23 Transmed Biotech Incorporated Methods and compositions for simultaneous analysis of multiple analytes
US5290707A (en) * 1991-11-25 1994-03-01 The United States Of America As Represented By The Secretary Of The Army Method for detection of microorganisms
WO1994010551A1 (en) 1992-10-30 1994-05-11 Sarasep, Inc. Method for particulate reagent sample treatment
US5429923A (en) 1992-12-11 1995-07-04 President And Fellows Of Harvard College Method for detecting hypertrophic cardiomyophathy associated mutations
US5367474A (en) 1993-02-08 1994-11-22 Coulter Corporation Flow cytometer
CA2125228A1 (en) * 1993-06-08 1994-12-09 Thomas J. Mercolino Three-color flow cytometry with automatic gating function
US5981180A (en) * 1995-10-11 1999-11-09 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and methods

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