Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin in the blood fibrinolytic system. It also plays an important role in the nervous system, including the processes of neuronal migration, neurite outgrowth, and neuronal plasticity. tPA has been suggested to have a role in several neuropathological conditions such as cerebral ischemia, seizures, and demyelinating diseases. The sensitive quantitative measurement of functionally active rat tPA in plasma and other biological fluid samples is easily performed with this 96 well strip format ELISA kit. The level of active tPA in normal rat plasma was found to be 1.6 ng/ml. The assay measures active tPA in the 0.1-50 ng/ml range. Samples giving rat tPA levels above 50 ng/ml should be diluted in blocking buffer before use. Samples of plasma in citrate or EDTA may be assayed with this kit. Plasma in heparin is not recommended. 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 tPA. If plasma samples were collected in citrate the pH should be brought up to neutral with the 10X TBS provided in the kit. Serum and cell culture media at neutral pH may also be used. Functionally active tPA will form a covalent complex with the biotinylated human PAI-1 which is bound to the avidin on the plate. Complexed tPA will not bind to the PAI-1 and will not be detected by the assay. After appropriate washing steps, anti-rat tPA 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 tPA in the samples. A standard calibration curve is prepared using dilutions of purified tPA and is measured along with the test samples. All reagents and standards are provided in these ELISA kits.

This assay uses an exclusive enzyme capture technology to only detect functionally active protein.

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