This is a Phase I proposal that is directed at the development of a rapid and quantitative diagnostic immunoassay for acute myocardial injury. This immunoassay will utilize a proven dry chemistry test system incorporating a cardiac-specific troponin peptide chemically conjugated to active-site inactivated coagulation factor Xa. The test principle involves competition between peptide- Xa and blood troponin for an insolublized anti-troponin monoclonal antibody in the first stage of the assay, followed by UV irradiation to reactivate the factor Xa that subsequently converts prothrombin to thrombin, which in the presence of fibrinogen results in a fibrin clot. The clotting time in this assay will be inversely proportional to the degree of oscillation of paramagnetic iron oxide particles in the dry chemistry test matrix.
The Specific Aims of this proposal are as follows. Firstly, Xa- troponin T peptide conjugates will be prepared and characterized. Conjugation will be acheived through amino groups on Xa and a C- terminal sulfhydryl group on the peptide using a heterobifunctional crosslinking agent that is capable of reacting with a free sulfhydryl group at one end and a free amino group at the other. In the second Specific Aim, the Applicant intends to develop standard calibration curves for the troponin immunoassay using blood samples spiked with known amounts of peptide or troponin. Next, the Applicant will develop a test card-based assay format capable of performing the troponin immunoassay on a single drop of blood using a modified TAS instrument retrofitted with a UV light source. Finally, the Applicant intends to validate the measurements against an established reference method using blood samples obtained from myocardial infarction patients.
