CA2227895A1 - Multiplexed analysis of clinical specimens apparatus and methods - Google Patents
Multiplexed analysis of clinical specimens apparatus and methodsInfo
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- CA2227895A1 CA2227895A1 CA002227895A CA2227895A CA2227895A1 CA 2227895 A1 CA2227895 A1 CA 2227895A1 CA 002227895 A CA002227895 A CA 002227895A CA 2227895 A CA2227895 A CA 2227895A CA 2227895 A1 CA2227895 A1 CA 2227895A1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
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- G—PHYSICS
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- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5094—Chemical 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
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- G01N2015/1477—Multiparameters
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- Y—GENERAL 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
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
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- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/973—Simultaneous 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 realtime while also reducing the cost of performing diagnostic and genetic assays.
Claims (85)
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 ... Cn) and sufficiently different in at least one of said classification parameters from beads in any other subset so that the profile of classification parameter values within each subset detectable by flow cytometry is unique;
(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 bead has been coupled is identifiable by flow cytometry based on the unique classification parameter profile of the beads.
(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 ... Cn) and sufficiently different in at least one of said classification parameters from beads in any other subset so that the profile of classification parameter values within each subset detectable by flow cytometry is unique;
(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 bead has been coupled is identifiable by flow cytometry based on the unique classification parameter 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, C3 ... Cn) and sufficiently different in at least one of said classification parameters from beads in any other subset so that the profile of classification parameter values within each subset detectable by flow cytometry is unique;
(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 subset identity and therefore the reactant to which the bead has been coupled is identifiable based on the unique classification parameter profile of the bead.
(a) the beads in each subset are sufficiently homogeneous with respect to at least three selected classification parameters (C1, C2, C3 ... Cn) and sufficiently different in at least one of said classification parameters from beads in any other subset so that the profile of classification parameter values within each subset detectable by flow cytometry is unique;
(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 subset identity and therefore the reactant to which the bead has been coupled is identifiable based on the unique classification parameter profile of the bead.
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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; 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 to be tested under conditions that will allow reactions between analytes of interest in the fluid and the reactants on the beads in said set, wherein a reaction between a reactant and an analyte of interest on a bead causes a change in the value of a fluorescent signal (Fm) emitted from said bead;
(c) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values; and (e) detecting the presence or absence of a particular analyte of interest in said sample as a function of the identification in step (d) and a change in the Fm values of the beads in each of said subsets in the reacted fluid sample relative to the Fm values of the beads in each of said subsets not reacted with said fluid.
(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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; 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 to be tested under conditions that will allow reactions between analytes of interest in the fluid and the reactants on the beads in said set, wherein a reaction between a reactant and an analyte of interest on a bead causes a change in the value of a fluorescent signal (Fm) emitted from said bead;
(c) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values; and (e) detecting the presence or absence of a particular analyte of interest in said sample as a function of the identification in step (d) and a change in the Fm values of the beads in each of said subsets in the reacted fluid sample relative to the Fm values of the beads in each of said subsets not reacted with said fluid.
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 .. . Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; 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 to be tested under conditions that will allow reactions between analytes of interest in the fluid and the reactants on the beads in said set;
(c) mixing with the reacted bead sample a fluorescent label under conditions such that said label will bind to and thereby increase the value of a fluorescent signal Fm emitted from said bead;
(d) analyzing the reacted sample containing the fluorescent label by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values; and (f) detecting the presence or absence of a particular analyte of interest in said sample as a function of the identification in step (e) and an increase in the Fm values of the beads in each of said subsets in the reacted fluid sample relative to the Fm values of the beads in each of said subsets not reacted with said fluid.
(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 .. . Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; 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 to be tested under conditions that will allow reactions between analytes of interest in the fluid and the reactants on the beads in said set;
(c) mixing with the reacted bead sample a fluorescent label under conditions such that said label will bind to and thereby increase the value of a fluorescent signal Fm emitted from said bead;
(d) analyzing the reacted sample containing the fluorescent label by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values; and (f) detecting the presence or absence of a particular analyte of interest in said sample as a function of the identification in step (e) and an increase in the Fm values of the beads in each of said subsets in the reacted fluid sample relative to the Fm values of the beads in each of said subsets not reacted with said fluid.
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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique, (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 labeled compound which competes with said analyte for reaction with said reactant;
(b) mixing, to produce a reacted bead sample, the beadset with the fluid to be tested under conditions that will allow reactions between analytes of interest in the fluid and the reactants on the beads in said set and thereby to allow the analytes to competitively inhibit or displace the fluorescently labeled compounds from said beads, resulting in a decrease in a fluorescent signal Fm emitted from a bead with which an analyte of interest in the fluid has reacted;
(c) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values; and (e) detecting the presence or absence of a particular analyte of interest in said sample as a function of the identification in step (d) and an increase in the Fm values of the beads in each of said subsets in the reacted fluid sample relative to the Fm values of the beads in each of said subsets not reacted with said fluid.
;'/
' ]' ' ';p
(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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique, (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 labeled compound which competes with said analyte for reaction with said reactant;
(b) mixing, to produce a reacted bead sample, the beadset with the fluid to be tested under conditions that will allow reactions between analytes of interest in the fluid and the reactants on the beads in said set and thereby to allow the analytes to competitively inhibit or displace the fluorescently labeled compounds from said beads, resulting in a decrease in a fluorescent signal Fm emitted from a bead with which an analyte of interest in the fluid has reacted;
(c) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values; and (e) detecting the presence or absence of a particular analyte of interest in said sample as a function of the identification in step (d) and an increase in the Fm values of the beads in each of said subsets in the reacted fluid sample relative to the Fm values of the beads in each of said subsets not reacted with said fluid.
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' ]' ' ';p
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 and 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 Fm signal 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 said syndrome 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 antibodies coupled to said beads in said set.
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 19 wherein said analyte comprises autoantibodies and said antigens comprise oligopeptide epitopes reactive with said autoantibodies, said fluorescent labels comprise fluorescent monoclonal antibodies reactive with said epitopes and wherein the presence of the analyte autoantibodies is detected as a result in a decrease of Fm.
27. The method of claim 3 wherein said analytes are enzymes, said reactants are fluorescently labeled substrates for said enzymes, said change in Fm results from cleavage of said substrates from said beads.
28. The method of claim 27 wherein said enzymes are selected from the groups consisting of proteases, glycosidases, nucleotidases, oxidoreductases, hydrolyases, esterases, convertases, ligases, transferases, phosphorylases, lyases, lipases, peptidases, dehydrogenases, oxidases, phospholipases, decarboxylases, invertases, aldolases, transminases, synthetases, and phosphotases.
29. The method of claim 3 wherein the fluid to be tested is selected from the group consisting of 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.
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 (C1, C2, C3 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to an immunoglobulin that corresponds to the immunoglobulin to be assayed for in the fluid sample;
(b) obtaining a fluorescently labeled immunoglobulin-binding reagent capable of reacting with the immunnglobulins to be detected;
(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample to be tested and the fluorescently labeled immunoglobulin-binding reagent under conditions that will allow competitive binding reactions between the immunoglobulin-binding reagent and immunoglobulin in the fluid to be tested and between the immunoglobulin-binding reagent and the immunoglobulin on the beads in said set, wherein a reaction between a bead-bound immunoglobulin and the fluorescently labeled immunoglobulin-binding reagent causes an increase in the value of a fluorescent signal (Fm) emitted from said bead;
(d) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values; and (f) detecting a corresponding immunoglobulin in said sample as a function of the identification in step (e) and a change in the Fm values of the beads in each of said subsets in the reacted fluid sample relative to the Fm values of the beads in each of said subsets not reacted with said fluid.
(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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to an immunoglobulin that corresponds to the immunoglobulin to be assayed for in the fluid sample;
(b) obtaining a fluorescently labeled immunoglobulin-binding reagent capable of reacting with the immunnglobulins to be detected;
(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample to be tested and the fluorescently labeled immunoglobulin-binding reagent under conditions that will allow competitive binding reactions between the immunoglobulin-binding reagent and immunoglobulin in the fluid to be tested and between the immunoglobulin-binding reagent and the immunoglobulin on the beads in said set, wherein a reaction between a bead-bound immunoglobulin and the fluorescently labeled immunoglobulin-binding reagent causes an increase in the value of a fluorescent signal (Fm) emitted from said bead;
(d) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values; and (f) detecting a corresponding immunoglobulin in said sample as a function of the identification in step (e) and a change in the Fm values of the beads in each of said subsets in the reacted fluid sample relative to the Fm values of the beads in each of said subsets not reacted with said fluid.
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 32 wherein said immunoglobulins to be detected are immunoglobulins belonging to different immunoglobulin sub-classes.
34. The method of claim 33 wherein said subclasses 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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to a monoclonal antibody preparation which is specific for an epitope that is the same epitope as that epitope which binds to an immunoglobulin to be assayed for;
(b) obtaining a plurality of fluorescently labeled reagents wherein each of said reagents bears an epitope to which the monoclonal antibody preparation coupled to the beads within a subset binds;
(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample to be tested and the fluorescently labeled reagents under conditions that will allow competitive binding reactions between the fluorescently labeled reagents and immunoglobulin in the fluid to be tested and between the fluorescently labeled reagents and the monoclonal antibodies on the beads wherein a reaction between a bead-bound antibody and the fluorescently labeled reagent causes an increase in the value of a fluorescent signal (Fm) emitted from said bead;
(d) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm value of each bead analyzed;
(e) identifying the subset to which each bead belongs and therefore the monoclonal antibody on the bead as a function of the unique profile of 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 a change in the Fm values of the beads in each of said subsets in the reacted fluid sample relative to the Fm~ values of the beads in each of said subsets not reacted with said fluid.
(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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to a monoclonal antibody preparation which is specific for an epitope that is the same epitope as that epitope which binds to an immunoglobulin to be assayed for;
(b) obtaining a plurality of fluorescently labeled reagents wherein each of said reagents bears an epitope to which the monoclonal antibody preparation coupled to the beads within a subset binds;
(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample to be tested and the fluorescently labeled reagents under conditions that will allow competitive binding reactions between the fluorescently labeled reagents and immunoglobulin in the fluid to be tested and between the fluorescently labeled reagents and the monoclonal antibodies on the beads wherein a reaction between a bead-bound antibody and the fluorescently labeled reagent causes an increase in the value of a fluorescent signal (Fm) emitted from said bead;
(d) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm value of each bead analyzed;
(e) identifying the subset to which each bead belongs and therefore the monoclonal antibody on the bead as a function of the unique profile of 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 a change in the Fm values of the beads in each of said subsets in the reacted fluid sample relative to the Fm~ values of the beads in each of said subsets not reacted with said fluid.
36. The method of claim 35 wherein the epitopes are epitopes located on viral antigens.
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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to an antibody which is specific for an analyte to be assayed for in the fluid sample;
(b) obtaining a plurality of preparations of antibody molecules wherein each of said preparations contains fluorescently labeled antibodies specific for an analyte to be assayed for in the fluid sample;
(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample to be tested and the fluorescently labeled antibodies under conditions that will allow binding reactions between the antibody that is coupled to the bead, the analyte of interest in the fluid to be tested, and the fluorescently labeled antibodies so as to bind said fluorescent antibodies to said beads though binding to said enzymes which are in turn bound to said beads though said bead-bound antibodies and wherein a bridging reaction between a bead-bound antibody, the analyte to which that antibody binds, and the fluorescently labeled antibody specific for said enzyme causes an increase in the value of a fluorescent signal (Fm) emitted from said bead;
(d) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values; and (f) detecting the analyte in said sample as a function of the identification in step (e) and a change in the Fm values of the beads in each of said subsets in the reacted fluid sample relative to values of the beads in each of said subsets not reacted with said fluid.
(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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to an antibody which is specific for an analyte to be assayed for in the fluid sample;
(b) obtaining a plurality of preparations of antibody molecules wherein each of said preparations contains fluorescently labeled antibodies specific for an analyte to be assayed for in the fluid sample;
(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample to be tested and the fluorescently labeled antibodies under conditions that will allow binding reactions between the antibody that is coupled to the bead, the analyte of interest in the fluid to be tested, and the fluorescently labeled antibodies so as to bind said fluorescent antibodies to said beads though binding to said enzymes which are in turn bound to said beads though said bead-bound antibodies and wherein a bridging reaction between a bead-bound antibody, the analyte to which that antibody binds, and the fluorescently labeled antibody specific for said enzyme causes an increase in the value of a fluorescent signal (Fm) emitted from said bead;
(d) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values; and (f) detecting the analyte in said sample as a function of the identification in step (e) and a change in the Fm values of the beads in each of said subsets in the reacted fluid sample relative to values of the beads in each of said subsets not reacted with said fluid.
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 the 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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to a peptide which provides a given epitope;
(b) obtaining a fluorescently labeled monoclonal antibody of interest;
(c) mixing, to produce a reacted bead sample, the beadset with the fluorescently labeled 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 a reaction between a bead-bound peptide and the fluorescently labeled monoclonal antibody causes an increase in the value of a fluorescent signal (Fm) emitted from said bead;
(d) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values;
and (f) detecting the particular epitope to which the monoclonal antibody binds as a function of the identification in step (e) and a change in the Fm values of the beads in each of said subsets in the sample relative to the Fm values of beads not reacted with said monoclonal antibody.
(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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to a peptide which provides a given epitope;
(b) obtaining a fluorescently labeled monoclonal antibody of interest;
(c) mixing, to produce a reacted bead sample, the beadset with the fluorescently labeled 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 a reaction between a bead-bound peptide and the fluorescently labeled monoclonal antibody causes an increase in the value of a fluorescent signal (Fm) emitted from said bead;
(d) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values;
and (f) detecting the particular epitope to which the monoclonal antibody binds as a function of the identification in step (e) and a change in the Fm values of the beads in each of said subsets in the sample relative to the Fm values of beads not reacted with said monoclonal antibody.
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 given 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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to an antigen derived from one of said pathogens of interest:
(b) obtaining a fluorescently labeled immmunoglobulin-reactive reagent;
(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample and the fluorescently labeled immunoglobulin-reactive reagent under conditions that will allow binding reactions between the bead-bound antigen and antibody in said sample and the fluorescently labeled immunoglobulin-reactive reagent wherein a reaction between a bead-bound antigen, antibody in said fluid sample and the fluorescently labeled reagent causes an increase in the value of a fluorescent signal (Fm) emitted from said bead;
(d) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values;
and (f) detecting the particular epitope to which the monoclonal antibody binds as a function of the identification in step (e) and a change in the Fm values of the beads in each of said subsets in the sample relative to the Fm values of beads not reacted with said fluid sample.
(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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to an antigen derived from one of said pathogens of interest:
(b) obtaining a fluorescently labeled immmunoglobulin-reactive reagent;
(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample and the fluorescently labeled immunoglobulin-reactive reagent under conditions that will allow binding reactions between the bead-bound antigen and antibody in said sample and the fluorescently labeled immunoglobulin-reactive reagent wherein a reaction between a bead-bound antigen, antibody in said fluid sample and the fluorescently labeled reagent causes an increase in the value of a fluorescent signal (Fm) emitted from said bead;
(d) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values;
and (f) detecting the particular epitope to which the monoclonal antibody binds as a function of the identification in step (e) and a change in the Fm values of the beads in each of said subsets in the sample relative to the Fm values of beads not reacted with said fluid sample.
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 labeled immunoglobulin-reactive reagent is anti-Human IgG.
45. The method of claim 43 wherein said fluorescently labeled 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 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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to an antigen derived from an allergen of interest;
(b) obtaining a fluorescently labeled IgE reactive reagent;
(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample and the fluorescently labeled reagent under conditions that will allow binding reactions between the bead-bound allergen and antibody in said sample and the fluorescently labeled IgE reactive reagent wherein a reaction between a bead-bound allergen, antibody in said fluid sample and the fluorescently labeled IgE-reactive reagent causes an increase in the value of a fluorescent signal (Fm) emitted from said bead;
(d) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm value of each bead analyzed;
(e) identifying the subset to which each bead belongs and therefore the allergen on said bead as a function of the unique profile of classification parameter values;
and (f) detecting the particular epitope to which the monoclonal antibody binds as a function of the identification in step (e) and a change in the Fm values of the beads in each of said subsets in the sample relative to the Fm values of beads not reacted with said fluid sample.
(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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to an antigen derived from an allergen of interest;
(b) obtaining a fluorescently labeled IgE reactive reagent;
(c) mixing, to produce a reacted bead sample, the beadset with the fluid sample and the fluorescently labeled reagent under conditions that will allow binding reactions between the bead-bound allergen and antibody in said sample and the fluorescently labeled IgE reactive reagent wherein a reaction between a bead-bound allergen, antibody in said fluid sample and the fluorescently labeled IgE-reactive reagent causes an increase in the value of a fluorescent signal (Fm) emitted from said bead;
(d) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm value of each bead analyzed;
(e) identifying the subset to which each bead belongs and therefore the allergen on said bead as a function of the unique profile of classification parameter values;
and (f) detecting the particular epitope to which the monoclonal antibody binds as a function of the identification in step (e) and a change in the Fm values of the beads in each of said subsets in the sample relative to the Fm values of beads not reacted with said fluid sample.
47. The method of claim 46 wherein said allergen 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 labeled IgE reactive reagent is anti-human IgE
49. The method of claim 46 wherein said fluorescently labeled IgE reactive reagent is anti-canine IgE.
50. A method of flow cytometric analysis capable of quantitating the concentration of an analyte of interest in a 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, ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to a reactant that will specifically react with the selected analyte of interest in the sample to be tested; 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 to be tested under conditions that will allow reactions between the analyte of interest in the fluid sample and the reactants on the beads in said set, wherein a reaction between a reactant and an analyte of interest on a bead causes a change in the value of a fluorescent signal (Fm) emitted from said bead;
(c) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm value of each bead analyzed;
(d) identifying the subset to which each bead belongs and therefore the concentration of reactant with which the bead was coupled as a function of the unique profile of classification parameter values; and (e) detecting the concentration of the analyte of interest in said sample as a function of the identification in step (d) and the Fm values of the beads in each of said subsets relative to the Fm 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.
(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, ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to a reactant that will specifically react with the selected analyte of interest in the sample to be tested; 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 to be tested under conditions that will allow reactions between the analyte of interest in the fluid sample and the reactants on the beads in said set, wherein a reaction between a reactant and an analyte of interest on a bead causes a change in the value of a fluorescent signal (Fm) emitted from said bead;
(c) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm value of each bead analyzed;
(d) identifying the subset to which each bead belongs and therefore the concentration of reactant with which the bead was coupled as a function of the unique profile of classification parameter values; and (e) detecting the concentration of the analyte of interest in said sample as a function of the identification in step (d) and the Fm values of the beads in each of said subsets relative to the Fm 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 the concentration of an analyte of interest in a 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, ...Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to a reactant that will specifically react with the analyte of interest in the sample to be tested; 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 a fluorescently labeled competitive inhibitor of the reaction between the analyte of interest and the reactant on the beads and with the fluid sample under conditions that will allow reactions between the analyte of interest in the fluid sample 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 the value of a fluorescent signal (Fm) emitted from said bead;
(c) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm value of each bead analyzed;
(d) identifying the subset to which each bead belongs and therefore the concentration of reactant with which the bead was coupled as a function of the unique profile of classification parameter values;
(e) assigning a bead subset value to each bead subset with 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 Fm 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 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.
(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, ...Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to a reactant that will specifically react with the analyte of interest in the sample to be tested; 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 a fluorescently labeled competitive inhibitor of the reaction between the analyte of interest and the reactant on the beads and with the fluid sample under conditions that will allow reactions between the analyte of interest in the fluid sample 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 the value of a fluorescent signal (Fm) emitted from said bead;
(c) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm value of each bead analyzed;
(d) identifying the subset to which each bead belongs and therefore the concentration of reactant with which the bead was coupled as a function of the unique profile of classification parameter values;
(e) assigning a bead subset value to each bead subset with 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 Fm 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 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 the concentration of an analyte 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 said subset;
(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3, ...Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to a reactant that will specifically react with a selected analyte of interest in a fluid to be tested; 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 with a fluorescently labeled 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 in the fluid and the reactants on the beads in said set, wherein a reaction between a reactant and an analyte of interest on a bead causes a decrease in the value of a fluorescent signal (Fm) emitted from said bead;
(c) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm value of each bead analyzed;
(d) identifying the subset to which each bead belongs and therefore the concentration of reactant with which the bead was coupled as a function of the unique profile of classification parameter values; and (e) assigning a bead subset value to each bead subset with correlates relatively with the concentration of analyte with which the bead subset was coupled; and (f) determining an inter-bead subset slope from a plot of mean Fm 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.
(a) obtaining a beadset comprising a plurality of subsets of beads wherein the beads in said subset;
(1) are sufficiently homogeneous with respect to each of at least three selected classification parameter (C1, C2, C3, ...Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique; and (2) are coupled to a reactant that will specifically react with a selected analyte of interest in a fluid to be tested; 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 with a fluorescently labeled 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 in the fluid and the reactants on the beads in said set, wherein a reaction between a reactant and an analyte of interest on a bead causes a decrease in the value of a fluorescent signal (Fm) emitted from said bead;
(c) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm value of each bead analyzed;
(d) identifying the subset to which each bead belongs and therefore the concentration of reactant with which the bead was coupled as a function of the unique profile of classification parameter values; and (e) assigning a bead subset value to each bead subset with correlates relatively with the concentration of analyte with which the bead subset was coupled; and (f) determining an inter-bead subset slope from a plot of mean Fm 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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique, (2) are coupled to a nucleic acid that will specifically hybridize with a selected nucleic acid analyte of interest in a fluid to be tested, (3) are reactive with a fluorescently labeled nucleic acid probe which competes with said nucleic acid analyte for hybridization with said nucleic acid coupled to the bead;
(b) mixing, to produce a reacted bead sample, the beadset with the fluid to be tested under conditions that will allow hybridization between nucleic acid analytes of interest in the fluid and the nucleic acids coupled to the beads in said beadset and thereby to allow the nucleic acid analytes in said fluid to inhibit hybridization between the fluorescently labeled nucleic acids with the nucleic acids coupled to said beads, resulting in a decrease in a fluorescent signal Fm emitted from a bead with which a nucleic acid analyte of interest in the fluid has reacted;
(c) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values; and (e) detecting the presence or absence of a particular analyte of interest in said sample as a function of the identification in step (d) and an increase in the Fm values of the beads in each of said subsets in the reacted fluid sample from the Fm values of the beads in each of said subsets not reacted with said fluid.
(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 ... Cn) values and sufficiently different from beads in any other subset in at least one of said classification parameter values so that the profile of classification parameter values within each subset detectable by flow cytometry is unique, (2) are coupled to a nucleic acid that will specifically hybridize with a selected nucleic acid analyte of interest in a fluid to be tested, (3) are reactive with a fluorescently labeled nucleic acid probe which competes with said nucleic acid analyte for hybridization with said nucleic acid coupled to the bead;
(b) mixing, to produce a reacted bead sample, the beadset with the fluid to be tested under conditions that will allow hybridization between nucleic acid analytes of interest in the fluid and the nucleic acids coupled to the beads in said beadset and thereby to allow the nucleic acid analytes in said fluid to inhibit hybridization between the fluorescently labeled nucleic acids with the nucleic acids coupled to said beads, resulting in a decrease in a fluorescent signal Fm emitted from a bead with which a nucleic acid analyte of interest in the fluid has reacted;
(c) analyzing the reacted sample by flow cytometry to determine the classification parameter value profile and an Fm 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 unique profile of classification parameter values; and (e) detecting the presence or absence of a particular analyte of interest in said sample as a function of the identification in step (d) and an increase in the Fm values of the beads in each of said subsets in the reacted fluid sample from the Fm values of the beads in each of said subsets not reacted with said fluid.
54. The method of claim 3 wherein said analytes are enzymes, said reactants are fluorescent molecules which upon reaction with the enzyme lose fluorescence, said change in Fm results from alteration of said substrates attached to said beads.
55. The method of claim 3 wherein said analytes are enzymes, said reactants are non-fluorescent molecules which upon reaction with the enzyme become fluorescent, and said change in Fm results from alteration of said substrates attached to said beads.
56. The method of claim 3 wherein said analytes are convertases which produce active enzymes from inactive precursors, said reactants are inactive precursors that are converted to active enzyme which in turn are reactants of fluorescently labeled substrates for said newly activated enzymes, and said change in Fm results from cleavage of said substrates from said beads.
57. The method of claim 5 wherein said analytes are enzymes, said reactants are molecules attached to a bead which, upon reaction with the enzyme, become ligates for a fluorescently labeled ligand, and wherein said change in Fm results from reaction of the new ligate with the fluorescently labeled ligand.
58. The method of claim 3 wherein said analyte is a cofactor which produces an active enzyme from an inactive apo-enzymes, said reactant is a fluorescently labeled substrate for said activated enzyme, and said change in Fm results from cleavage of said substrate from said active enzyme.
59. A method of processing a plurality of data signals generated by a flow cytometer in real-time, each said data signal being associated with a specific cytometric target and encoding a forward light scatter value, a side light scatter value, a red fluorescence value, an orange fluorescence value, and a green fluorescence value, comprising:
(a) receiving a data signal;
(b) extracting from said data signal (1) a forward light scatter value, (2) a side light scatter value, (3) a red fluorescence value, (4) an orange fluorescence value, and (5) a green fluorescence value;
(c) classifying said cytometric target into one of a plurality of classes, referred to as an identified class, said classification being a function of said extracted (1) forward light scatter value, (2) side light scatter value, (3) red fluorescence value, and (4) orange fluorescence value;
(d) incrementing a class-count value associated with said identified class, said class-count value encoding the number of cytometric targets classified as belonging to said identified class;
(e) accumulating a green-fluorescence-sum value associated with said identified class, said green fluorescence sum-value encoding an arithmetic sum of said extracted green fluorescence value for all cytometric targets classified as belonging to said identified class;
(f) repeating the operations described in paragraphs (a) through (e) for subsequent data signals;
(g) generating, for each of said plurality of classes, one or more outcome-description signals encoding textual information correlated with the class-count value and with the green-fluorescence-sum value for said class; and (h) displaying said textual information.
(a) receiving a data signal;
(b) extracting from said data signal (1) a forward light scatter value, (2) a side light scatter value, (3) a red fluorescence value, (4) an orange fluorescence value, and (5) a green fluorescence value;
(c) classifying said cytometric target into one of a plurality of classes, referred to as an identified class, said classification being a function of said extracted (1) forward light scatter value, (2) side light scatter value, (3) red fluorescence value, and (4) orange fluorescence value;
(d) incrementing a class-count value associated with said identified class, said class-count value encoding the number of cytometric targets classified as belonging to said identified class;
(e) accumulating a green-fluorescence-sum value associated with said identified class, said green fluorescence sum-value encoding an arithmetic sum of said extracted green fluorescence value for all cytometric targets classified as belonging to said identified class;
(f) repeating the operations described in paragraphs (a) through (e) for subsequent data signals;
(g) generating, for each of said plurality of classes, one or more outcome-description signals encoding textual information correlated with the class-count value and with the green-fluorescence-sum value for said class; and (h) displaying said textual information.
60. The method of claim 59 wherein said specific cytometric target is an appropriately labeled bead.
61. The method of claim 60 wherein each one of said plurality of classes is associated with one bead subset, said bead subset formed in accordance with claim 2.
62. The method of claim 59 wherein the operation of paragraph ~ is further comprised of performing a reasonableness test on said cytometric target's identified class, said reasonableness test being a function of one or more of said identified class' (1) forward light scatter value, (2) side light scatter value, (3) red fluorescence value, and (4) orange fluorescence value.
63. A method of processing a plurality of data signals generated by a flow cytometer, each said data signal being associated with a specific flow cytometric target and encoding a plurality of classification parameter values and one or more measurement parameter values, comprising:
(a) receiving a data signal;
(b) extracting said plurality of classification parameter values and said one or more measurement parameter values from said data signal;
(c) classifying said cytometric target into one of a plurality of classes, referred to as an identified class, said classification being a function of said plurality of extracted classification parameter values;
(d) incrementing a class-count value associated with said identified class;
(e) accumulating each of said one or more extracted measurement values into one or more respective accumulation-values for said identified class;
(f) repeating the operations described in paragraphs (a) through (e) for subsequent data signals;
(g) generating, for each of said plurality of classes, one or more outcome-description signals encoding information correlated with the class-count and with the one or more accumulation-sum values for said class; and (h) displaying said textual information.
(a) receiving a data signal;
(b) extracting said plurality of classification parameter values and said one or more measurement parameter values from said data signal;
(c) classifying said cytometric target into one of a plurality of classes, referred to as an identified class, said classification being a function of said plurality of extracted classification parameter values;
(d) incrementing a class-count value associated with said identified class;
(e) accumulating each of said one or more extracted measurement values into one or more respective accumulation-values for said identified class;
(f) repeating the operations described in paragraphs (a) through (e) for subsequent data signals;
(g) generating, for each of said plurality of classes, one or more outcome-description signals encoding information correlated with the class-count and with the one or more accumulation-sum values for said class; and (h) displaying said textual information.
64. The method of claim 63 wherein said processing in performed in real-time.
65. The method of claim 64 wherein each one of said plurality of classes is associated with one bead subset, said bead subset formed in accordance with claim 2.
66. The method of claim 63 wherein said specific cytometric target is an appropriately labeled bead.
67. The method of claim 63 wherein said data signal encodes a plurality of classification parameter values selected from the group consisting of forward light scatter, side light scatter, red fluorescence, and orange fluorescence.
68. The method of claim 63 wherein said data signal encodes one or more measurement parameter values selected from the group consisting of orange fluorescence and green fluorescence.
69. The method of claim 63 wherein said one or more outcome-description signals encodes textual information.
70. The method of claim 63 wherein each of said one or more outcome-description signals is determined by either an OVER-UNDER test or a SHIFT test.
71. An machine readable assay database, stored in a storage device, for the processing of flow-cytometric measurement data comprising:
(a) an assay definition table, said assay definition table encoding (1) one or more measurement subset token identifiers, (2) for each subset token identifier, one or more baseline measurement parameter values, and (3) for each subset token identifier, an interpretation test-type token;
(b) a discriminant function table, said discriminant function table encoding a classification decision tree based on one or more classification measurement parameters, said one or more classification measurement parameters encoded in said flow-cytometric measurement data;
(c) an interpretation table, said interpretation table encoding textual assay outcome-description information; and (d) a results table, said results table capable of encoding statistical accumulation of real-time flow-cytometric measurement data.
(a) an assay definition table, said assay definition table encoding (1) one or more measurement subset token identifiers, (2) for each subset token identifier, one or more baseline measurement parameter values, and (3) for each subset token identifier, an interpretation test-type token;
(b) a discriminant function table, said discriminant function table encoding a classification decision tree based on one or more classification measurement parameters, said one or more classification measurement parameters encoded in said flow-cytometric measurement data;
(c) an interpretation table, said interpretation table encoding textual assay outcome-description information; and (d) a results table, said results table capable of encoding statistical accumulation of real-time flow-cytometric measurement data.
72. A method of processing a plurality of data signals, in real-time, generated by a diagnostic device, each of said plurality of data signals being associated with a specific diagnostic target and encoding a plurality of classification parameter values and one or more measurement parameter values, comprising:
(a) receiving a data signal;
(b) extracting said plurality of classification parameter values and said one or more measurement parameter values from said data signal;
(c) classifying said diagnostic target into one of a plurality of classes, referred to as an identified class, said classification being a function of said plurality of extracted classification parameter values;
(d) incrementing a class-count value associated with said identified class;
(e) accumulating each of said one or more extracted measurement values into one or more respective accumulation-values for said identified class;
(f) repeating the operations described in paragraphs (a) through (e) for subsequent data signals;
(g) generating, for each of said plurality of classes, one or more outcome-description signals encoding information correlated with the class-count and with the one or more accumulation-sum values for said class; and (h) displaying said outcome-description signals.
(a) receiving a data signal;
(b) extracting said plurality of classification parameter values and said one or more measurement parameter values from said data signal;
(c) classifying said diagnostic target into one of a plurality of classes, referred to as an identified class, said classification being a function of said plurality of extracted classification parameter values;
(d) incrementing a class-count value associated with said identified class;
(e) accumulating each of said one or more extracted measurement values into one or more respective accumulation-values for said identified class;
(f) repeating the operations described in paragraphs (a) through (e) for subsequent data signals;
(g) generating, for each of said plurality of classes, one or more outcome-description signals encoding information correlated with the class-count and with the one or more accumulation-sum values for said class; and (h) displaying said outcome-description signals.
73. The method of claim 72 wherein said diagnostic device is selected from the group consisting of a flow cytometer and a cell sorter.
74. A program storage device that is readable by a computer, said program storage device having encoded therein a program of instructions that includes instructions for executing the method steps of a specified one of claims 59, 63, 71, and 72.
75. A method for flow cytometric analysis to detect genetic mutations in a DNA comprising:
(a) obtaining beads coupled to an oligonucleotide molecule designed to hybridize with a selected PCR product of interest;
(b) mixing the beads with said PCR product under conditions that will allow hybridization between said PCR product and the oligonucleotide coupled to the beads and thereby to allow the PCR product to inhibit hybridization between a fluorescently labeled nucleic acid probe that is completely complementary to said oligonucleotide coupled to said beads, (c) adding said fluorescent probe to the mixture;
(d) analyzing the reacted sample by flow cytometry to determine the fluorescence of each bead analyzed; and (e) detecting the genetic mutation or absence thereof as a result of the degree of fluorescence on the beads.
(a) obtaining beads coupled to an oligonucleotide molecule designed to hybridize with a selected PCR product of interest;
(b) mixing the beads with said PCR product under conditions that will allow hybridization between said PCR product and the oligonucleotide coupled to the beads and thereby to allow the PCR product to inhibit hybridization between a fluorescently labeled nucleic acid probe that is completely complementary to said oligonucleotide coupled to said beads, (c) adding said fluorescent probe to the mixture;
(d) analyzing the reacted sample by flow cytometry to determine the fluorescence of each bead analyzed; and (e) detecting the genetic mutation or absence thereof as a result of the degree of fluorescence on the beads.
76. A method to detect a genetic mutations in a DNA comprising:
(a) obtaining beads coupled to an oligonucleotide molecule, said oligonucleotide molecule designed to hybridize with a selected PCR product of interest;
(b) mixing said beads with said PCR product, under conditions that will allow hybridization between said PCR product and the oligonucleotide coupled to the beads, to form a reacted mixture;
(c) adding a fluorescent probe to said reacted mixture;
(d) determining the fluorescence of the beads by flow cytometry; and (e) detecting the genetic mutation, or absence thereof, as a result of the degree of the determined fluorescence on the beads.
(a) obtaining beads coupled to an oligonucleotide molecule, said oligonucleotide molecule designed to hybridize with a selected PCR product of interest;
(b) mixing said beads with said PCR product, under conditions that will allow hybridization between said PCR product and the oligonucleotide coupled to the beads, to form a reacted mixture;
(c) adding a fluorescent probe to said reacted mixture;
(d) determining the fluorescence of the beads by flow cytometry; and (e) detecting the genetic mutation, or absence thereof, as a result of the degree of the determined fluorescence on the beads.
77. A method of detecting a genetic mutation in a DNA comprising the steps of:
(a) selecting an oligonucleotide probe for said genetic mutation;
(b) preparing a fluorescent DNA probe complementary to the oligonucleotide probe coupling said selected probe to each one of a plurality of beads to form a bead aliquot;
(c) selecting PCR primers to amplify a region of said DNA corresponding to said selected probe;
(d) amplifying said genetic mutation by PCR to form PCR products;
(e) mixing said bead aliquot, said PCR products and said fluorescent probe to form a mixture;
(f) incubating said mixture to promote under competitive hybridization conditions;
(g) measuring the fluorescence said beads by flow cytometry; and (h) detecting said genetic mutation, or absence thereof, as a function of the measured fluorescence of said beads.
(a) selecting an oligonucleotide probe for said genetic mutation;
(b) preparing a fluorescent DNA probe complementary to the oligonucleotide probe coupling said selected probe to each one of a plurality of beads to form a bead aliquot;
(c) selecting PCR primers to amplify a region of said DNA corresponding to said selected probe;
(d) amplifying said genetic mutation by PCR to form PCR products;
(e) mixing said bead aliquot, said PCR products and said fluorescent probe to form a mixture;
(f) incubating said mixture to promote under competitive hybridization conditions;
(g) measuring the fluorescence said beads by flow cytometry; and (h) detecting said genetic mutation, or absence thereof, as a function of the measured fluorescence of said beads.
78. The method of claim 77 wherein said genetic mutation is selected from the group consisting of mutations in MEN2a, MEN2b, MEN1, ret proto-oncogene, LDL receptor, NF1, NF type 2, BRCA1, BRCA2, BRCA3, APC, adenosine deaminase, XPAC, ERCC6 excision repair gene, fmrl, Duchenne's muscular dystrophy gene, myotonic dystrophy protein kinase, androgen receptor, Huntington's, HPRT, apolipoprotein E, HEXA, steroid 2-hydroxylase, angiotensin, hNMLH1, 2 mismatch repair, APC, Rb, p53, bcr/abl, bcl-2 gene, chromosomes 11 to 14 and chromosomes 15 to 17 gene transpositions, and genes encoding ion transporters.
79. The method of claim 77 wherein said oligonucleotide probe has a length of between 5 and 500 nucleotides.
80. The method of claim 77 wherein said PCR primers are designed to amply a region of said DNA corresponding to said oligonucleotide probe.
81. The method of claim 77 wherein said fluorescent probe is selected from the group consisting of DNA sequences to wild-type or mutant sequences coupled to the beads.
82. A kit for detection of a genetic mutation in a DNA comprising:
(a) a first container comprising beads coupled to an oligonucleotide designed to hybridize with a selected PCR product of interest;
(b) a second container a PCR primer designed to amplify a section of DNA complementary to said oligonucleotide; and (c) a third container comprising a fluorescent labeled DNA probe capable of selectively hybridizing said oligonucleotide.
(a) a first container comprising beads coupled to an oligonucleotide designed to hybridize with a selected PCR product of interest;
(b) a second container a PCR primer designed to amplify a section of DNA complementary to said oligonucleotide; and (c) a third container comprising a fluorescent labeled DNA probe capable of selectively hybridizing said oligonucleotide.
83. The kit of claim 82, wherein said genetic mutation is selected from the group consisting of mutations in MEN2a, MEN2b, MEN1, ret proto-oncogene, LDL receptor, NF1, NF
type 2, BRCA1, BRCA2, BRCA3, APC, adenosine deaminase, XPAC, ERCC6 excision repair gene, fmr1, Duchenne's muscular dystrophy gene, myotonic dystrophy protein kinase, androgen receptor, Huntington's, HPRT, apolipoprotein E, HEXA, steroid 2-hydroxylase, angiotensin, hNMLH1, 2 mismatch repair, APC, Rb, p53, bcr/abl, bcl-2 gene, chromosomes 11 to 14 and chromosomes 15 to 17 gene transpositions, and genes encoding ion transporters.
type 2, BRCA1, BRCA2, BRCA3, APC, adenosine deaminase, XPAC, ERCC6 excision repair gene, fmr1, Duchenne's muscular dystrophy gene, myotonic dystrophy protein kinase, androgen receptor, Huntington's, HPRT, apolipoprotein E, HEXA, steroid 2-hydroxylase, angiotensin, hNMLH1, 2 mismatch repair, APC, Rb, p53, bcr/abl, bcl-2 gene, chromosomes 11 to 14 and chromosomes 15 to 17 gene transpositions, and genes encoding ion transporters.
84. The kit of claim 82 wherein said fluorescent labeled DNA probe has a length of between 5 and 500 nucleotides.
85. The method claim 3 where the analytes of interest are DNA segments, the reactant on the bead are DNA segment capable of specifically hybridizing to said analytes, and the fluorescent label is a fluorescent DNA segment also capable of specifically hybridizing with said reactant to compete with the hybridization of said reactant to said label.
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- 1996-10-10 AU AU73989/96A patent/AU7398996A/en not_active Abandoned
- 1996-10-10 AT AT96936310T patent/ATE496288T1/en not_active IP Right Cessation
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Cited By (1)
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CN111537426A (en) * | 2013-03-15 | 2020-08-14 | 贝克曼考尔特公司 | System and method for designing panels in flow cytometry |
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EP0852004A2 (en) | 1998-07-08 |
US6939720B2 (en) | 2005-09-06 |
DE69638321D1 (en) | 2011-03-03 |
CA2227895C (en) | 2012-07-17 |
ATE496288T1 (en) | 2011-02-15 |
AU7398996A (en) | 1997-04-30 |
EP0852004B1 (en) | 2011-01-19 |
US6524793B1 (en) | 2003-02-25 |
WO1997014028A3 (en) | 1997-09-25 |
US20050118574A1 (en) | 2005-06-02 |
WO1997014028A2 (en) | 1997-04-17 |
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