IMUBIND® Factor VIIa ELISA

Cat#873

INTENDED USE

The IMUCLONE™ Total TAFI ELISA is an in vitro assay for the measurement of TAFI antigen in human plasma, or in any fluid where TAFI may be present.

EXPLANATION OF THE TEST

TAFI, Thrombin Activatable Fibrinolytic Inhibitor, (also known as carboxypeptidase U and plasma pro-carboxypeptidase B) is a 60,000 D molecular weight glycoprotein (proenzyme form) present in human plasma1 that modulates fibrinolysis in vivo. This proenzyme is converted to a 35,000 D molecular ratio active form, TAFIa, following proteolytic cleavage by the thrombin/thrombomodulin complex. TAFIa possesses carboxypeptidase activity with a preference for cleaving lysine and arginine residues from the c-terminus of proteins. Modulation of fibrinolysis occurs when TAFIa cleaves C-terminal arginine and lysine residues of partially degraded fibrin.2,4,5 The removal of the c-terminus arginine and lysine residues from fibrin inhibits the continued degradation of fibrin by tPA activated plasmin.3

Plasma also contains carboxypeptidase N (CPN), which has enzymatic activity similar to that of TAFI. The total carboxypeptidase activity in plasma is found to be TAFI + CPN. TAFI, but not CPN, is inhibited by potato tuber carboxypeptidase inhibitor (PTCI). The ability of PTCI to selectively inhibit TAFI is used to determine that portion of the total carboxypeptidase activity contributed by TAFI.

TAFI may play a central role in thrombosis and fibrinolysis due to its ability to retard fibrin clot lysis.

PRINCIPLE OF THE PROCEDURE

Diluted plasma samples, biological fluid or TAFI Calibrator are added to microwells precoated with a murine monoclonal antibody specific for human TAFI. The antibody captures the TAFI antigen present in the solutions during an incubation period. Following a washing step, a goat anti-human TAFI polyclonal antibody coupled to horseradish peroxidase (HRP) is added to the microwells and binds to the captured TAFI antigen. Following another washing step, the peroxidase substrate Tetramethylbenzidine (TMB), in the presence of hydrogen peroxide, is added to the wells and the subsequent reaction yields a blue colored solution. Addition of sulfuric acid stops the reaction and turns the solution color yellow. The absorbance of the solution is measured at 450 nm. The absorbance is directly proportional to the amount of TAFI present in the sample.

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