First the biotinylated PAI-1 binds to the avidin coated wells. Next, active tPA present in the standard or unknown, complexes 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. Sample Types: biological fluids. Range: 0.1 - 50 ng/ml. Sensitivity: 0.1 ng/ml. Time: 2.5 hours ? Tissue-type Plasminogen Activator (tPA) is a member of the serine proteinase 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.