Active human tPA functional assay ELISA kit
Tissue plasminogen activator (tPA) is a serine protease that catalyzes the activation of plasminogen to plasmin. Clinical studies have indicated that high tPA levels may increase the risk for thrombosis, whereas decreased levels may cause neuronal plasticity and degeneration. The sensitive quantitative measurement of functionally active human tPA in plasma and other biological fluid samples is easily performed with this 96 well strip format ELISA kit. The basal level of tPA in healthy humans was found to be between 0.2-2 IU/ml. The assay measures active tPA in the 0-1 IU/ml range. Samples giving human tPA levels above 1 IU/ml should be diluted in blocking buffer before use. Samples of human 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 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 diluent 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. This kit cross reacts with rat, pig, and dog tPA and does not cross react with mouse tPA. After appropriate washing steps, polyclonal anti-human tPA primary antibody binds to the captured enzyme. Excess antibody is washed away and bound antibody is reacted with peroxidase conjugated secondary antibody. 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.
The Human tPA standard provided in this kit is calibrated against the WHO International Standard for Human tPA (NIBSC Code 98/714).