Urokinase plasminogen activator (uPA) is a serine protease that activates plasminogen to plasmin in the blood fibrinolytic system. It is also implicated in events related to cell invasion/migrations. The sensitive quantitative measurement of functionally active rat uPA in plasma and other biological fluid samples is easily performed with this 96 well strip format ELISA kit. The assay measures active uPA in the 0.05-10 ng/ml range. Samples giving rat uPA levels above 10 ng/ml should be diluted in blocking buffer before use. It is important to ensure a platelet free preparation of plasma as platelets can release PAI-1, which in turn could potentially form a complex with active uPA. If plasma samples were collected in citrate the pH should be brought up to neutral with 10X TBS. Serum and cell culture media at neutral pH may also be used. If using kidney extracts that have been extracted using triton X, dialyze to remove the triton X before using in the assay. Detergents such as triton X may interfere with the assay. Functionally active uPA will form a covalent complex with the biotinylated human PAI-1 which is bound to the avidin on the plate. Inactive or complexed uPA will not bind to the PAI-1 and will not be detected by the assay. After appropriate washing steps, anti-rat uPA primary antibody binds to the captured enzyme. Excess antibody is washed away, and bound antibody is reacted with the secondary antibody conjugated to HRP. Following an additional washing step, TMB substrate is used for color development at 450 nm. Color development is proportional to the concentration of active uPA in the samples. A standard calibration curve is prepared using dilutions of purified uPA and is measured along with the test samples. All reagents and standards are provided in these ELISA kits.

Suggested additional reagents: 10X Wash Buffer, TMB Substrate, Avidin Plate, Secondary Antibody