Background

Tissue-type Plasminogen Actvator (tPA) is a member of the serine protease family. tPA functions to lyse fibrin clots into soluble plasmin fragments. tPA is active in two forms, single chain and two chain. The two chain tPA is created via interaction with the plasmin product cleaving the single chain. This two chain form is regarded as the more active form. Both single chain and two chain tPA can complex with PAI-1. PAI-1 acts as an inhibitor for tPA by binding to the tPA and thus stifling its ability to lyse fibrin. tPA can serve as an indicator of both myocardial infarction for patients with impaired fibrinolytic systems as well as a marker for type-II diabetes.

Assay Principle

This is an ELISA for the quantitative analysis of active tPA levels in biological fluid. This test kit operates on the basis of sandwich ELISA where free, active, tPA enzyme complexes with PAI-1 and is quantified with the use of an HRP labeled secondary antibody.

Provided is biotinylated PAI-1 bound to avidin coated wells. This allows active tPA present in the standard or unknown, to complex with PAI-1. Inactive or complexed tPA is removed in a subsequent wash step. A primary antibody specific for tPA is then added to each well followed by the HRP conjugated secondary antibody. The bound conjugated secondary antibody is detected by the addition of substrate, which generates an optimal color after 10 minutes. Quantitative test results may be obtained by the measure and comparison of the sample and standard absorbance readings when read with a microplate reader at 450 nm