Multiple antigen detection assays and reagents

• US 20050095627A1
• Filed: 09/03/2004
• Published: 05/05/2005
• Est. Priority Date: 09/03/2003
• Status: Abandoned Application

• Current Assignee: Salk Institute for Biological Studies
• Original Assignee: Salk Institute for Biological Studies
• Inventors: Kolman, John L., Wahl, Geoffrey M.
• Application Number:
  US10/933,671
• Publication Number:
  US 20050095627A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Provided herein are multiple antigen detection (MAD) assays and reagents for performing such assays. MAD assay embodiments are useful for identifying proximal antigens in a sample (e.g., antigens existing in a complex), and are performed by contacting a sample with two or more hybrids, where each hybrid comprises a nucleic acid and a binding partner that specifically binds to an antigen. In these embodiments, the hybrid nucleic acids hybridize to one another when the hybrids bind to proximal antigens, and the hybridization product then is detected, often by a polymerase chain reaction (PCR) process or a nucleic acid binding agent comprising a detectable label. MAD assays and reagents are applicable to antigen detection in situ, chromatin immunoprecipitation and genomic sequencing, detection of proximal intramolecular regions in an antigen, detecting antigen multimerization, diagnostics and therapeutics. Also provided are kits for conducting MAD applications.

Claims

1. A method for detecting a first molecule and a second molecule in a sample, which comprises:

contacting a sample with a first hybrid, which comprises a first binding partner and a first nucleic acid, and a second hybrid, which comprises a second binding partner and a second nucleic acid, wherein;

the first binding partner specifically binds to the first molecule and the second binding partner specifically binds to the second molecule,

the first nucleic acid comprises a first nucleotide sequence complementary to a second nucleotide sequence in the second nucleic acid and is capable of forming a hybridization product with the second nucleic acid when the first hybrid is bound to the first molecule and the second hybrid is bound to the second molecule and the first molecule and the second molecule are in proximity;

diluting the sample and the hybrids, wherein the molecules and hybrids are not in association with a solid phase;

extending the hybridization product to form an extension product after diluting the sample and the hybrids; and

identifying the presence or absence of the extension product, whereby identifying the extension product detects the first molecule and the second molecule in the sample.

2. The method of claim 1, wherein the first molecule and second molecule are independently selected from the group consisting of a non-hybrid nucleic acid, a protein, a protein in association with another protein, and a protein in association with a non-hybrid nucleic acid.

3. The method of claim 1, which comprises contacting the sample with a cross-linking agent before contacting the sample with the hybrids.

4. The method of claim 1, wherein the first nucleotide sequence and second nucleotide sequence are six or fewer nucleotides in length.

5. The method of claim 1, which comprise contacting the system with a third nucleic acid, wherein the third nucleic acid hybridizes to a single-stranded region in the first nucleic acid or second nucleic acid in the hybridization product and abuts the 3′

end of the other of the first nucleic acid or second nucleic acid when hybridized.

6. The method of claim 5, wherein there is no gap between the 5′

end of the third nucleic acid and the 3′

end of the first nucleic acid or second nucleic acid.

7. The method of claim 1, wherein the first binding partner, second binding partner, or first binding partner and second binding partner is an antibody or antibody fragment.

8. The method of claim 1, wherein the presence or absence of the extension product is identified by contacting the system with polymerase chain reaction (PCR) reagents and detecting the presence or absence of PCR products.

9. A method for detecting a first molecule and a second molecule in a sample, which comprises:

contacting a sample with a first hybrid, which comprises a first binding partner and a first nucleic acid, and a second hybrid, which comprises a second binding partner and a second nucleic acid, wherein;

the first binding partner specifically binds to the first molecule and the second binding partner specifically binds to the second molecule,

the first nucleic acid comprises a first nucleotide sequence complementary to a second nucleotide sequence in the second nucleic acid and is capable of forming a hybridization product with the second nucleic acid when the first hybrid is bound to the first molecule and the second hybrid is bound to the second molecule and the first molecule and the second molecule are in proximity;

optionally extending the hybridization product to form an extension product;

contacting the hybridization product or extension product with a nucleic acid binding agent that specifically binds to a nucleotide sequence in the hybridization product or extension product; and

identifying the presence or absence of the nucleic acid binding agent bound to the hybridization product or extension product, whereby identifying the nucleic acid binding agent bound to the hybridization product or extension product detects the first molecule and the second molecule in the sample.

10. The method of claim 9, wherein the nucleic acid binding agent comprises a nucleic acid binding protein or a fragment thereof that specifically binds to a nucleotide sequence in the hybridization product or extension product.

11. The method of claim 10, wherein the nucleic acid binding agent comprises a lac repressor protein, a gal4 protein, a tus protein, or a fragment thereof.

12. The method of claim 9, wherein the nucleic acid binding agent comprises a detectable label.

13. The method of claim 9, wherein one or more of the hybrids are in association with a solid phase.

14. A composition which comprises a first hybrid, which comprises a first binding partner and a first nucleic acid, and a second hybrid, which comprises a second binding partner and a second nucleic acid, wherein:

the first nucleic acid comprises a first nucleotide sequence complementary to a second nucleotide sequence in the second nucleic acid and is capable of forming a hybridization product with the second nucleic acid; and

the first nucleotide sequence and second nucleotide sequence are six or fewer nucleotides in length.

15. The composition of claim 14, wherein the first binding partner, the second binding partner, or the first binding partner and second binding partner are an antibody or antibody fragment.

16. A composition which comprises:

a first hybrid, which comprises a first binding partner and a first nucleic acid, and a second hybrid, which comprises a second binding partner and a second nucleic acid, wherein the first nucleic acid comprises a first nucleotide sequence complementary to a second nucleotide sequence in the second nucleic acid and is capable of forming a hybridization product with the second nucleic acid; and

a third nucleic acid capable of hybridizing to a single-stranded region of the first nucleic acid or second nucleic acid in the hybridization product, wherein the 5′

end of the third nucleic acid abuts the 3′

end of the other of the first nucleic acid or second nucleic acid when hybridized.

17. The composition of claim 16, wherein the first binding partner, the second binding partner, or the first binding partner and second binding partner are an antibody or antibody fragment.

18. A composition which comprises:

a first hybrid, which comprises a first binding partner and a first nucleic acid, and a second hybrid, which comprises a second binding partner and a second nucleic acid, wherein the first nucleic acid comprises a first nucleotide sequence complementary to a second nucleotide sequence in the second nucleic acid and is capable of forming a hybridization product with the second nucleic acid; and

a nucleic acid binding agent that specifically binds to a nucleotide sequence in the hybridization product or a nucleotide sequence in an extension product produced by extending the hybridization product.

19. The composition of claim 18, wherein the first binding partner, the second binding partner, or the first binding partner and second binding partner are an antibody or antibody fragment.

20. The composition of claim 18, wherein the nucleic acid binding agent comprises a lac repressor protein, a gal4 protein, a tus protein, or a fragment thereof that specifically binds to a nucleotide sequence in the hybridization product or extension product.

21. The composition of claim 18, wherein the nucleic acid binding agent comprises one or more components selected from the group consisting of a detectable label, a cytotoxic agent and a solid phase.

22. A method for detecting a molecule in a sample, which comprises:

contacting a sample with a first hybrid, which comprises a first binding partner and a first nucleic acid, and a second hybrid, which comprises a second binding partner and a second nucleic acid, wherein;

the first binding partner specifically binds to a first binding region in the molecule and the second binding partner specifically binds to a second binding region in the molecule,

the first nucleic acid comprises a first nucleotide sequence complementary to a second nucleotide sequence in the second nucleic acid and is capable of forming a hybridization product with the second nucleic acid when the first hybrid is bound to the first binding region and the second hybrid is bound to the second binding region;

diluting the sample and the hybrids, wherein the molecule and hybrids are not in association with a solid phase;

extending the hybridization product to form an extension product after diluting the sample and hybrids; and

identifying the presence or absence of the extension product, whereby identifying the extension product detects the molecule in the sample.

23. A method for detecting a molecule in a sample, which comprises:

contacting a sample with a first hybrid, which comprises a first binding partner and a first nucleic acid, and a second hybrid, which comprises a second binding partner and a second nucleic acid, wherein;

the first binding partner specifically binds to a first binding region in the molecule and the second binding partner specifically binds to a second binding region in the molecule,

the first nucleic acid comprises a first nucleotide sequence complementary to a second nucleotide sequence in the second nucleic acid and is capable of forming a hybridization product with the second nucleic acid when the first hybrid is bound to the first binding region and the second hybrid is bound to the second binding region;

optionally extending the hybridization product to form an extension product;

contacting the hybridization product or extension product with a nucleic acid binding agent that specifically binds to the nucleotide sequence of the hybridization product or extension product; and

identifying the presence or absence of the nucleic acid binding agent bound to the hybridization product or extension product, whereby identifying the nucleic acid binding agent bound to the hybridization product or extension product detects the molecule in the sample.