Universal shotgun assay

• US 20050221363A1
• Filed: 03/31/2005
• Published: 10/06/2005
• Est. Priority Date: 04/01/2004
• Status: Abandoned Application

• Current Assignee: Rules-Based Medicine, Inc.
• Original Assignee: Rules-Based Medicine, Inc.
• Inventors: Spain, Michael D., Chandler, Mark B.
• Application Number:
  US11/094,366
• Publication Number:
  US 20050221363A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: G01N 33/536 with immune complex formed in liquid phase
• CPC Class Codes: G01N 33/541 Double or second antibody , i.e. precipitating antibody
• CPC Class Codes: G01N 33/54333 Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
• CPC Class Codes: G16H 50/50 for simulation or modelling of medical disorders
• CPC Class Codes: G16H 50/70 for mining of medical data, e.g. analysing previous cases of other patients
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

A method for the multiplexed diagnosis of a plurality of different biomolecules in a fluid sample substantially simultaneously is provided. In accordance with a method of the invention, a substantial fraction of biomolecules in a fluid sample are complexed with a universal label and a secondary labeling reagent. Flow cytometric measurements may be used to identify and quantify, in real-time, by detecting the secondary reagent and universal label present in any of said complexes. The inventive technology enables the simultaneous, and automated, detection and interpretation of multiple biomolecules while also reducing the cost of performing diagnostic and genetic assays.

Claims

1. A method of revealing the identities of a plurality of different analytes present in a fluid sample comprising:

(a) contacting a fluid sample, which is suspected of harboring a plurality of different analytes, with a universal reagent capable of binding to all or substantially all of said plurality of different analytes, under conditions effective to label all or substantially all of said plurality of different analytes with said universal reagent;

(b) contacting said plurality of different analytes labeled with said universal reagent with a collection of distinguishable subpopulations of secondary reagent, each secondary reagent of a given distinguishable subpopulation being designed to interact selectively with a given analyte of said plurality of different analytes, under conditions effective to provide a plurality of complexes, each complex comprised of said distinguishable secondary reagent and said given analyte labeled with said universal reagent; and

(c) detecting through said universal reagent the existence of a complex and determining through said distinguishable secondary reagent the nature of said given analyte found therein, thereby revealing the identities of the plurality of different analytes present in the fluid sample.

2. The method of claim 1 in which the quantities of the plurality of different analytes in the fluid sample is revealed.

3. The method of claim 1 in which said universal reagent comprises one or more analyte binding moieties and one or more detectable labels.

4. The method of claim 3 in which the detectable label comprises a fluorescent group, a radioactive atom, or a combination thereof.

5. The method of claim 3 in which the analyte binding moiety is a protein, nucleic acid, or a lipid.

6. The method of claim 1 in which said universal reagent comprises a metal complex.

7. The method of claim 6 in which said metal complex comprises a platinum metal complex labeled with a fluorescent group.

8. The method of claim 6 in which said metal complex comprises a metalloporphyrin or platinum metal complex including (i) biotin or a biotin-containing moiety and (ii) a leaving group.

9. The method of claim 1 in which said collection of distinguishable subpopulations of secondary reagent comprises planar substrates, magnetic beads, or fluorescent microspheres.

10. The method of claim 1 in which said secondary reagent comprises polypeptides, polynucleotides, or combinations thereof.

11. The method of claim 10 in which said polypeptides comprise antibodies or the binding regions thereof.

12. The method of claim 9 in which said fluorescent microspheres form distinguishable subpopulations through distinguishable combinations of two or more fluorophores.

13. The method of claim 1 in which step (c) is carried out in a flow analyzer.

14. The method of claim 4 in which the universal reagent and the secondary reagent each exhibit one or more characteristic fluorescence emission classification parameters.

15. The method of claim 14 in which the universal reagents differ from the secondary reagents in an intensity of at least one fluorescence emission classification parameter.

16. The method of claim 1 in which said given analyte comprises a hormone, antigen, cytokine, antibody, or oligopeptide.

17. The method of claim 1 in which said given analyte comprises genomic DNA, cDNA, oligonucleotides, RNA, or RNA nucleotides.

18. The method of claim 1 in which the fluid sample is selected from the group consisting of plasma, serum, tears, mucus, saliva, urine, pleural fluid, spinal fluid, gastric fluid, sweat, semen, vaginal secretion, broncioalveolar lavage, sputum, breath condensate, peritoneal fluid, joint fluid, fluid from ulcers and other surface eruptions, blisters, abscesses, and extracts of tissues including biopsies of normal, malignant, or suspect tissues.

19. The method of claim 14 further comprising amplifying the fluorescence emission of the universal reagent or the secondary reagent.

20. The method of claim 1 in which the fluid sample comprises cell lysate.

21. The method of claim 1 in which the fluid sample is obtained from a mammal.

22. The method of claim 21 in which the mammal is a human.

23. The method of claim 1 further comprising removing unbound secondary reagent prior to performing step (c).

24. The method of claim 1 in which said universal reagent or said secondary reagent is coupled to a solid support.

25. The method of claim 24 in which the solid support comprises a microsphere, microchip, biochip, or planar array.

26. A method of revealing the identities of a plurality of different analytes present in a fluid sample comprising:

(a) contacting a fluid sample, which is suspected of harboring a plurality of different analytes, with a universal reagent capable of binding to all or substantially all of said plurality of different analytes, under conditions effective to label all or substantially all of said plurality of different analytes with said universal reagent;

(b) contacting said plurality of different analytes labeled with said universal reagent with a collection of distinguishable subpopulations of secondary reagent, each secondary reagent of a given distinguishable subpopulation being designed to interact selectively with a given analyte of said plurality of different analytes, under conditions effective to provide a plurality of complexes, each complex comprised of said distinguishable secondary reagent and said given analyte labeled with said universal reagent;

(c) contacting said plurality of complexes with excess reporter reagent, the reporter reagent having an affinity for at least a portion of said universal reagent and further comprised of a detectable label or cleavable group, under conditions effective to provide a plurality of complexes, each complex bearing a detectable label or cleavable group; and

(d) detecting through said detectable label the existence of a complex and determining through said distinguishable secondary reagent the nature of said given analyte found therein, thereby revealing the identities of the plurality of different analytes present in the fluid sample.

27. The method of claim 26 in which said universal reagent comprises a metal complex.

28. The method of claim 27 in which said metal complex comprises a platinum metal complex including biotin or a biotin-containing moiety.

29. The method of claim 28 in which the reporter reagent is avidin bound to a detectable label.

30. The method of claim 29 in which said detectable label gives rise to fluorescence, chemiluminescence, or a color change.

31. The method of claim 26, further comprising removing unbound reporter reagent prior performing step (d).

32. A method of arriving at a likelihood that a subject is suffering from or will later develop one or more diseases, comprising:

(a) contacting a fluid sample, which is suspected of harboring a plurality of different analytes, with a universal reagent capable of binding to all or substantially all of said plurality of different analytes, under conditions effective to label all or substantially all of said plurality of different analytes with said universal reagent;

(b) contacting said plurality of different analytes labeled with said universal reagent with a collection of distinguishable subpopulations of secondary reagent, each secondary reagent of a given distinguishable subpopulation being designed to interact selectively with a given analyte of said plurality of different analytes, under conditions effective to provide a plurality of complexes, each complex comprised of said distinguishable secondary reagent and said given analyte labeled with said universal reagent;

(c) contacting said plurality of complexes with excess reporter reagent, the reporter reagent having an affinity for at least a portion of said universal reagent and further comprised of a detectable label or cleavable group, under conditions effective to provide a plurality of complexes, each complex bearing a detectable label or cleavable group;

(d) detecting through said detectable label the existence of a complex and determining through said distinguishable secondary reagent the nature of said given analyte found therein, thereby revealing the identities and quantities of the plurality of different analytes present in the fluid sample;

(e) comparing analyte data generated from step (d) with a database of accumulated analyte data, which accumulated analyte date comprises a compilation of analyte data obtained from a statistically significant number of subjects suffering from or who later develop one or more diseases;

(f) optionally arriving at a likelihood that such subject is suffering from or will later develop one or more diseases based, at least in part, on the results of said comparison.

33. A method of generating an animal model of a disease comprising:

(a) contacting a fluid sample, which is suspected of harboring a plurality of different analytes, with a universal reagent capable of binding to all or substantially all of said plurality of different analytes, under conditions effective to label all or substantially all of said plurality of different analytes with said universal reagent;

(b) contacting said plurality of different analytes labeled with said universal reagent with a collection of distinguishable subpopulations of secondary reagent, each secondary reagent of a given distinguishable subpopulation being designed to interact selectively with a given analyte of said plurality of different analytes, under conditions effective to provide a plurality of complexes, each complex comprised of said distinguishable secondary reagent and said given analyte labeled with said universal reagent;

(c) contacting said plurality of complexes with excess reporter reagent, the reporter reagent having an affinity for at least a portion of said universal reagent and further comprised of a detectable label or cleavable group, under conditions effective to provide a plurality of complexes, each complex bearing a detectable label or cleavable group;

(d) detecting through said detectable label the existence of a complex and determining through said distinguishable secondary reagent the nature of said given analyte found therein, thereby revealing the identities of the plurality of different analytes present in the fluid sample;

(e) determining a relationship between one or more given analytes and the disease of the population of subjects whose accumulated biochemical analyte data share similar features; and

(f) genetically manipulating an animal to express the one or more given analytes related to the disease of interest.

34. The method of claim 33 in which the animal is a rodent.

35. A method of determining the effects of drug administration on a subject expressing one or more given analytes comprising:

(a) contacting fluid samples taken from a subject both before and after administration of a drug, which samples are suspected of harboring a plurality of different analytes, with a universal reagent capable of binding to all or substantially all of said plurality of different analytes, under conditions effective to label all or substantially all of said plurality of different analytes with said universal reagent;

(b) contacting said plurality of different analytes labeled with said universal reagent with a collection of distinguishable subpopulations of secondary reagent, each secondary reagent of a given distinguishable subpopulation being designed to interact selectively with a given analyte of said plurality of different analytes, under conditions effective to provide a plurality of complexes, each complex comprised of said distinguishable secondary reagent and said given analyte labeled with said universal reagent;

(c) contacting said plurality of complexes with excess reporter reagent, the reporter reagent having an affinity for at least a portion of said universal reagent and further comprised of a detectable label or cleavable group, under conditions effective to provide a plurality of complexes, each complex bearing a detectable label or cleavable group;

(d) detecting through said detectable label the existence of a complex and determining through said distinguishable secondary reagent the nature of said given analyte found therein, thereby revealing the identities of the plurality of different analytes present in the fluid samples; and

(e) comparing the analyte data generated from the sample taken before administration of the drug with the analyte data generated from the sample taken after administration of the drug to provide the effect(s), if any, of said drug administration on the analyte data expressed by the subject.

36. A computer implemented method for generating information on a disease comprising:

(a) contacting a fluid sample, which is suspected of harboring a plurality of different analytes, with a universal reagent capable of binding to all or substantially all of said plurality of different analytes, under conditions effective to label all or substantially all of said plurality of different analytes with said universal reagent;

(b) contacting said plurality of different analytes labeled with said universal reagent with a collection of distinguishable subpopulations of secondary reagent, each secondary reagent of a given distinguishable subpopulation being designed to interact selectively with a given analyte of said plurality of different analytes, under conditions effective to provide a plurality of complexes, each complex comprised of said distinguishable secondary reagent and said given analyte labeled with said universal reagent;

(c) contacting said plurality of complexes with excess reporter reagent, the reporter reagent having an affinity for at least a portion of said universal reagent and further comprised of a detectable label, under conditions effective to provide a plurality of complexes, each complex bearing a detectable label or cleavable group;

(d) detecting through said detectable label the existence of a complex and determining through said distinguishable secondary reagent the nature of said given analyte found therein, thereby revealing the identities of the plurality of different analytes present in the fluid sample; and

(e) compiling analyte data generated from step (d) in a database.

37. The method of claim 36 in which said database is in a computer readable format.

39. The method of claim 37 which further comprises selecting a set of given analytes the presence or absence of which appears to correlate with a likelihood that a subject is suffering from or will later develop one or more diseases.

40. The method of claim 37 in which all the subjects chosen for inclusion in the database suffer from or will later develop the same or similar such one or more diseases.

38. A method of generating information useful in the creation of a diagnostic assay for arriving at a likelihood that a subject is suffering from or will later develop one or more diseases comprising:

(a) contacting a fluid sample obtained from a subject suffering from or who later develops one or more diseases, which fluid sample is suspected of harboring a plurality of different analytes, with a universal reagent capable of binding to all or substantially all of said plurality of different analytes, under conditions effective to label all or substantially all of said plurality of different analytes with said universal reagent;

(b) contacting said plurality of different analytes labeled with said universal reagent with a collection of distinguishable subpopulations of secondary reagent, each secondary reagent of a given distinguishable subpopulation being designed to interact selectively with a given analyte of said plurality of different analytes, under conditions effective to provide a plurality of complexes, each complex comprised of said distinguishable secondary reagent and said given analyte labeled with said universal reagent;

(c) detecting through said detectable label the existence of a complex and determining through said distinguishable secondary reagent the nature of said given analyte found therein, thereby revealing the identities of the plurality of different analytes present in the fluid sample;

(d) repeating steps (a) to (c) using fluid samples from a sufficient number of subjects suffering from or who later develop one or more diseases to provide a database of accumulated analyte data comprising a compilation of analyte data obtained from a statistically significant number of subjects suffering from or who later develop one or more diseases;

(e) extracting information from said database, which information is useful in the creation of a diagnostic assay for arriving at a likelihood that a subject is suffering from or will later develop one or more diseases.