Sensitive, multiplexed diagnostic assays for protein analysis

• US 6,489,116 B2
• Filed: 01/04/2001
• Issued: 12/03/2002
• Est. Priority Date: 01/24/2000
• Status: Expired due to Term

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Phylos, Inc. (Bristol-Myers Squibb Company)
• Inventors: Wagner, Richard W.
• Primary Examiner(s): Guzo, David
• Assistant Examiner(s): Leffers, Jr., Gerald G.
• Application Number:
  US09/755,663
• Publication Number:
  US 20010024784A1
• Time in Patent Office: 698 Days
• Field of Search: 435/6, 435/29, 435/320.1, 435/68.1, 435/69.1, 536/23.1, 536/23.4, 536/24.33, 536/24.3, 436/536, 436/518, 530/350, 530/300, 530/402
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors

Abstract

Disclosed herein are methods for detecting multiple compounds in a sample, involving: (a) contacting the sample with a mixture of binding reagents, the binding reagents being nucleic acid-protein fusions, each having (i) a protein portion which is known to specifically bind to one of the compounds and (ii) a nucleic acid portion which encodes the protein portion and which includes a unique identification tag; (b) allowing the protein portions of the binding reagents and the compounds to form complexes; (c) capturing the binding reagent-compound complexes; (d) amplifying the nucleic acid portions of the complexed binding reagents; and (e) detecting the unique identification tag of each of the amplified nucleic acids, thereby detecting the corresponding compounds in the sample. Also disclosed herein are kits for carrying out such methods.

Claims

1. A method for detecting multiple different compounds in a sample, said method comprising:

(a) providing both a sample containing different compounds and a mixture of binding reagents, said binding reagents comprising nucleic acid-protein fusions, each fusion comprising (i) a protein portion which is known to specifically bind to one of said compounds and (ii) a nucleic acid portion which encodes said protein portion and which includes a unique identification tag;

(b) contacting said sample with said binding reagents under conditions allowing said protein portions of said binding reagents and said compounds to form binding reagent-compound complexes;

(c) capturing the binding reagent-compound complexes;

(d) amplifying said unique identification tag included on said nucleic acid portions of said binding reagent compound complexes; and

(e) detecting said unique identification tag of each of said binding reagent- compound complexes, thereby detecting the corresponding compounds in said sample.

2. The method of claim 1, wherein said sample is a biological sample.

3. The method of claim 1, wherein said nucleic acid-protein fusion is an RNA-protein fusion.

4. The method of claim 1, wherein said nucleic acid-protein fusion is covalently linked through a peptide acceptor.

5. The method of claim 4, wherein said peptide acceptor is puromycin.

6. The method of claim 1, wherein said binding reagents do not bind said compounds through compound-specific antibody domains.

7. The method of claim 1, wherein each of said binding reagents comprises a scaffold domain.

8. The method of claim 7, wherein each of said binding reagents comprises a fibronectin scaffold domain.

9. The method of claim 8, wherein said fibronectin scaffold domain is the 10^th domain of fibronectin type III.

10. The method of claim 7, wherein each of said binding reagents comprises an antibody scaffold domain.

11. The method of claim 1, wherein said binding reagents bind said compounds with equilibrium constants of less than about 500 nM.

12. The method of claim 1, wherein said unique identification tags are detected using a solid support to which are immobilized nucleic acids specific for said unique identification tags and said detection is accomplished by hybridization of said unique identification tags to said immobilized nucleic acids.

13. The method of claim 1, wherein said amplifying step (d) is carried out using quantitative PCR.

14. The method of claim 1, wherein said compounds are proteins.

15. The method of claim 1, wherein said mixture of binding reagents comprises at least 5 different nucleic acid-protein fusions, each of said nucleic acid- protein fusions specifically binding to a different compound.

16. The method of claim 15, wherein said mixture of binding reagents comprises at least 100 different nucleic acid-protein fusions, each of said nucleic acid-protein fusions specifically binding to a different compound.

17. The method of claim 16, wherein said mixture of binding reagents comprises at least 40,000 different nucleic acid-protein fusions, each of said nucleic acid-protein fusions specifically binding to a different compound.

18. The method of claim 17, wherein said mixture of binding reagents comprises at least 500,000 different nucleic acid-protein fusions, each of said nucleic acid-protein fusions specifically binding to a different compound.

19. A kit for compound detection, said kit comprising:

(a) a nucleic acid-protein fusion, wherein the protein portion of said fusion specifically binds said compound and the nucleic acid portion of said fusion encodes said protein portion and includes a unique identification tag;

(b) a PCR primer pair, wherein the first primer hybridizes to said nucleic acid portion of said fusion 5′

to said unique identification tag and the second primer hybridizes to said nucleic acid portion of said fusion 3′

to said unique identification tag and hybridization of said first and second primers to said nucleic acid portion permits amplification of said unique identification tag; and

(c) a solid support comprising a nucleic acid which can hybridize to said unique identification tag.

20. The kit of claim 19, wherein said kit further comprises Taq polymerase.

21. The kit of claim 19, wherein said nucleic acid-protein fusion is an RNA-protein fusion.

22. The kit of claim 19, wherein said nucleic acid-protein fusion is covalently linked through a peptide acceptor.

23. The kit of claim 22, wherein said peptide acceptor is puromycin.

24. The kit of claim 19, wherein said nucleic acid-protein fusion does not bind said compound through a compound-specific antibody domain.

25. The kit of claim 19, wherein said nucleic acid-protein fusion comprises a scaffold domain.

26. The kit of claim 25, wherein said nucleic acid-protein fusion comprises a fibronectin scaffold domain.

27. The kit of claim 26, wherein said fibronectin scaffold domain is the 10^th domain of fibronectin type III.

28. The kit of claim 25, wherein said nucleic acid-protein fusion comprises an antibody scaffold domain.

29. The kit of claim 19, wherein said nucleic acid-protein fusion binds said compound with an equilibrium constant of less than about 500 nM.

30. The kit of claim 19, wherein said solid support is a chip.

31. The kit of claim 19, wherein said solid support comprises an ordered array of single-stranded nucleic acids on its surface, each of said single-stranded nucleic acids being capable of hybridizing to a different unique identification tag.

32. The kit of claim 19, wherein said compound is a protein.

33. The kit of claim 19, wherein said kit comprises at least 5 different nucleic acid-protein fusions, each different nucleic acid-protein fusion characterized as being capable of specific binding to a different compound.

34. The kit of claim 33, wherein said kit comprises at least 100 different nucleic acid-protein fusions, each different nucleic acid-protein fusion characterized as being capable of specific binding to a different compound.

35. The kit of claim 34, wherein said kit comprises at least 40,000 different nucleic acid-protein fusions, each different nucleic acid-protein fusion characterized as being capable of specific binding to a different compound.

36. The kit of claim 35, wherein said kit comprises at least 500,000 different nucleic acid-protein fusions, each specifically binding to a different compound.