Detection of biomarkers and biomarker complexes

1. A method of sequentially detecting three epitopes of a biomarker in a biological sample, said method comprising:

(a) contacting said biological sample with a device having an immobilized binding agent on the surface thereof in a pattern that generates a signal so that said biomarker in said sample specifically binds to said immobilized binding agent,

(b) contacting said surface with a first detecting binding agent, wherein said first detecting binding agent specifically binds to a first epitope of the biomarker and not to a second or third epitope of the biomarker;

(c) detecting said first epitope by a change in signal produced by said pattern upon binding of said first detecting binding agent to said first epitope;

(d) contacting said surface with a second detecting binding agent, wherein said second detecting binding agent specifically binds to the second epitope of said biomarker and does not specifically bind to said first or third epitope;

(e) detecting said second epitope by a change in signal produced by said pattern upon binding of said second detecting binding agent to said second epitope;

(f) contacting said surface with a third detecting binding agent, wherein said third detecting binding agent specifically binds to the third epitope of said biomarker and does not specifically bind to said first or second epitope; and

(g) detecting said third epitope by a change in signal produced by said pattern upon binding of said third detecting binding agent to said third epitope.

2. The method of claim 1 , wherein at least one of said first, second, or third binding agents detects a posttranslational modification, alternative splicing, or degradation.

3. The method of claim 1 , wherein the biomarker is a component of cardiac troponin.

4. The method of claim 1 , further comprising repeating steps (a)-(g) with a second biological sample.

5. The method of claim 4 , wherein the second biological sample is from a different subject than the biological sample.

6. The method of claim 4 , wherein the second biological sample is from the same subject as the biological sample.

7. The method of claim 6 , wherein the second biological sample is obtained a predetermined period of time after the biological sample.

8. The method of claim 4 , wherein the second biological sample is from a different tissue than the biological sample.

9. The method of claim 1 , wherein steps (b), (d), and (f) occur simultaneously.

10. The method of claim 1 , wherein steps (b), (d), and (f) occur sequentially.

11. The method of claim 1 , wherein the first, second, and third detecting binding agents are enzyme-labeled antibodies, and enzyme substrates are added one at a time to detect the first, second, and third epitopes.

12. The method of claim 1 , wherein the biomarker is a protein, a nucleic acid, a lipid, a toxin, a drug, a virus, or a cell.

13. The method of claim 2 , wherein the posttranslational modification is an addition of a functional group, an addition of a protein or a peptide, a change in the chemical nature of an amino acid, or a structural change.

14. The method of claim 2 , wherein the posttranslational modification is phosphorylation, glycosylation, acetylation, alkylation, formylation, oxidation, biotinylation, ubiquitination, deamination, demethylation, or proteolytic cleavage.

15. The method of claim 1 , wherein said immobilizing binding agent is an anti-TnI antibody, an anti-TnT antibody, or an anti-TnC antibody.

16. The method of claim 1 , wherein said first detecting binding agent is an anti-TnI antibody, an anti-TnT antibody, or an anti-TnC antibody.

17. The method of claim 16 , wherein said anti-TnI antibody is anti-cTnI antibody; said anti-TnT antibody is anti-cTnT antibody; or said anti-TnC antibody is anti-cTnC antibody.

18. The method of claim 1 , wherein said signal is diffraction of light illuminating said device.