Non-competitive immunoassays to detect small molecules using nanopeptamers

Patent metadata

Field	Value
Publication number	US-10101324-B2
Application number	US-201314648242-A
Country	US
Kind code	B2
Filing date	Nov 26, 2013
Priority date	Dec 3, 2012
Publication date	Oct 16, 2018
Grant date	Oct 16, 2018

Abstract

A method for noncompetitive detection of a small analyte using nanopeptamers, and devices useful for performing the methods. Nanopeptamers include a self-associating oligomeric protein that is attached to peptides that bind to an immune complex between the target analyte and a capture antibody. The noncompetitive methods allow for the direct detection of small analytes with increased sensitivity over competitive methods directed to the same target analyte, and provide a positive readout which is useful for rapid tests and on-site detection of small analytes such as such as pesticides, persistent organic pollutants, explosives, toxins, medicinal and abused drugs, and hormones.

First claim

What is claimed is:

1. A method for non-competitive detection of a small analyte, the method comprising:
(a) contacting at least one immune complex comprising an antibody specifically bound to the analyte, with an affinity agent comprising a self-associated oligomeric protein displaying multiple copies of a peptide, wherein at least one copy of the peptide specifically binds to the immune complex, and the peptide comprises from about 5 to about 50 amino acids; and
(b) detecting the bound affinity agent, thereby detecting the analyte, where the analyte has a molecular weight of less than about 2500 daltons.

2. The method of claim 1 , wherein the self-associated oligomeric protein is streptavidin, avidin, or verotoxin.

3. The method of claim 1 , wherein the self-associated oligomeric protein is conjugated to a detectable label.

4. The method of claim 3 , wherein the detectable label is an enzyme, a fluorescent label, a dye, or a magnetic particle.

5. The method of claim 1 , wherein the peptide specifically binds to the immune complex.

6. The method of claim 1 , wherein the peptide comprises from about 5 to about 25 amino acids.

7. The method of claim 1 , wherein the peptide is obtained from a combinatorial biological or synthetic peptide library by selection with the analyte-antibody immune complex.

8. The method of claim 1 , wherein the analyte has a molecular weight of less than about 1000 daltons.

9. The method of claim 1 , wherein the analyte has a molecular weight of less than about 750 daltons.

10. The method of claim 1 , wherein the analyte has a molecular weight of less than about 500 daltons.

11. The method of claim 1 , further comprising contacting a sample suspected of containing the analyte with the antibody that specifically binds to the analyte, thereby forming the immune complex.

12. The method of claim 1 , wherein the peptide is non-covalently attached to the self-associated oligomeric protein.

13. The method of claim 1 , wherein the peptide is covalently attached to the self-associated oligomeric protein.

14. The method of claim 1 , wherein the self-associated oligomeric protein comprises a plurality of subunit monomers each linked to a copy of the peptide.

15. The method of claim 14 , wherein each subunit monomer is linked to the peptide by a peptide bond.

16. The method of claim 14 , wherein each subunit monomer is linked to the peptide by a spacer comprising at least one amino acid.

17. A device for detecting a small analyte, the device comprising: (a) a solid support comprising an antibody that specifically binds to the analyte immobilized thereon; and (b) an affinity agent comprising a self-associated oligomeric protein displaying multiple copies of a peptide, the peptide comprising from about 5 to about 50 amino acids, wherein each peptide is capable of specifically binding to an immune complex formed when the antibody binds to the analyte, where the analyte has a molecular weight of less than about 2500 daltons.

18. The device of claim 17 , wherein the self-associated oligomeric protein is conjugated to a detectable label.

19. The device of claim 18 , wherein the detectable label is detectable by the human eye.

20. The device of claim 19 , wherein the detectable label is a dye.

21. The device of claim 17 , wherein the affinity agent is immobilized on the solid support.

Assignees

• Univ California
• Univ De La Republica Facultad De Quimica

Inventors

• Gonzalez-Sapienza Gualberto
• Hammock Bruce
• Gonzalez-Techera Andres
• Vanrell Lucia
• Lassabe Gabriel

Classifications

• G01N33/54306 Primary

  Solid-phase reaction mechanisms · CPC title