Analyte detection
The present disclosure provides methods and/or kits for detecting an analyte in a sample. Some embodiments provide a method for detecting a non-nucleic acid analyte in a sample using a solid substrate comprising a bound immobilization agent and an antibody capture agent and a detectable agent, which can bind to the analyte. The antibody capture agent comprises, at a plurality of sites, a ligand for the immobilization agent. A complex between the analyte, the antibody capture agent and a detectable agent is formed and immobilized on the solid substrate by binding between the immobilization agent and the ligand. In some embodiments, the ligand and the immobilization agent are a binding pair comprising a peptide tag and an anti-peptide tag antibody.
1. A method for detecting an analyte, comprising: covalently binding an antibody capture agent with a plurality of peptide tags to form a tagged antibody capture agent;
forming an analyte complex comprising the analyte bound to the tagged antibody capture agent and to a detectable agent;
introducing the analyte complex to an anti-peptide antibody immobilized on a substrate;
immobilizing the analyte complex to the substrate by binding at least one of the plurality of peptide tags with the anti-peptide antibody; and
detecting the analyte by detection of the detectable agent.
2. The method according to claim 1 , wherein the analyte is a non-nucleic acid analyte.
3. The method according to claim 1 , wherein the tagged antibody capture agent complexes with a non-nucleic acid portion of the analyte.
4. The method according to claim 1 , wherein the tagged antibody capture agent binds with the analyte substantially exclusive of nucleic acid-nucleic acid interactions.
5. The method according to claim 1 , wherein at least one of the plurality of peptide tags is not naturally occurring.
6. The method according to claim 1 , wherein the covalently binding comprises cross-linking.
7. The method according to claim 6 , wherein the cross-linking comprises covalent conjugation by primary amines in the antibody capture agent.
8. The method according to claim 1 , wherein the tagged antibody capture agent is in solution.
9. The method according to claim 8 , wherein the solution has a tagged antibody capture agent concentration in the range from 50 to 500 ng/ml.
10. The method according to claim 1 , wherein the tagged antibody capture agent has a dissociation constant, Kd, of between 10 −6 and 10 −12 M.
11. The method according to claim 1 , wherein the detectable agent is in solution.
12. The method according to claim 1 , wherein the detectable agent comprises a detectable tag.
13. The method according to claim 12 , wherein the detectable tag comprises one or more of an enzyme, a fluorophore, and a lanthanide ion.
14. The method according to claim 1 , wherein the anti-peptide antibody is bound to the substrate via a non-covalent attachment.
15. The method according to claim 1 , wherein the substrate is at least partially blocked.
16. The method according to claim 1 , wherein the complexing occurs in a solution.
17. The method according to claim 16 , wherein the solution has a tagged antibody capture agent concentration in the range from 50 to 500 ng/ml.
18. The method according to claim 1 , wherein the analyte is provided in a biological sample.
19. The method according to claim 18 , wherein the sample has an analyte concentration of 100 ng/ml or less.
20. The method according to claim 18 , wherein the sample comprises one or more proteins separate from the analyte wherein the one or more proteins have a total concentration of more than 0.01 mg/ml.
21. The method according to claim 19 , wherein the sample comprises one or more proteins separate from the analyte wherein the one or more proteins have a total concentration of more than 0.01 mg/ml.
22. The method according to claim 18 , wherein the sample comprises one or more of blood, serum, urine, milk, cell lysate, and a derivative thereof.
23. The method according to claim 21 , wherein the sample comprises one or more of blood, serum, urine, milk, cell lysate, and a derivative thereof.
24. The method according to claim 1 , wherein the complexing and the immobilizing occur in a single reaction vessel, and more than one analyte is detected in the single reaction vessel.
25. The method according to claim 1 , wherein the complexing occurs in a first reaction vessel and the immobilizing occurs in a second reaction vessel.
26. The method according to claim 1 , wherein the immobilizing comprises incubating.
27. The method according to claim 26 , wherein the analyte is detected in less than 120 minutes after the incubating is initiated.
28. The method according to claim 1 , further comprising one or more washings after immobilizing.
29. The method according to claim 28 , wherein the one or more washings consists of no more than one washing.
30. The method according to claim 1 , wherein the plurality of peptide tags comprises a peptide comprising the amino acid sequence DYKDDDDK (SEQ ID NO. I).