Background

Urokinase-type Plasminogen Actvator (uPA) is a glycosylated serine protease that is synthesized in endothelial and kidney epithelial cells. There are two forms of uPA, the low molecular weight (LMW ~31 kDa) and high molecular weight (HMW ~55 kDa). The HMW form undergoes autoproteolysis resulting in the LMW plus and 18.5 kDa amino terminal fragment (ATF). It is this ATF that has been shown to inhibit proliferation and invasion of cancer cells by binding to uPA receptors (1). 

Assay Principle

This is an ELISA for the quantitative analysis of active uPA levels in biological fluid. This test kit operates on the basis of sandwich ELISA where free active uPA enzyme complexes with PAI-1 and is quantitated 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 uPA is removed in a subsequent wash step. A primary antibody specific for uPA 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.