IMUBIND® Factor VIIa ELISA

Cat#827

INTENDED USE

The IMUBIND® Factor VIIa ELISA is an enzyme-linked immunoassay for the quantitation of activated human factor VII (FVIIa) in plasma as well as in cell culture supernatants. This ELISA detects FVIIa as well as FVIIa complexed with Tissue Factor (TF/fVIIa). The assay is limited to research use only. It is not for use in diagnostic procedures.

EXPLANATION OF THE TEST

Factor VII (FVII) is the first zymogen of the extrinsic pathway of blood coagulation. Activation of FVII occurs via cleavage of the proenzyme by proteases (e.g. factors IXa, Xa, XIIa and thrombin). Factor VII is also subject to auto-activation by Factor VIIa (FVIIa). The FVIIa molecule is the result of enzymatic cleavage at the Arg152-Ile153 bond. It consists of a 36,000 Dalton heavy chain and a 22,000 Dalton light chain held together by a disulfide bond. When FVIIa complexes with Tissue Factor, an enhanced enzymatic complex is formed that rapidly promotes coagulation. Tissue Factor Pathway Inhibitor (TFPI) negatively regulates the activity of the TF/FVIIa complex.

FVIIa levels in plasma are approximately 5 ng/mL, 1% of FVII.

PRINCIPLE OF THE PROCEDURE

The IMUBIND FVIIa ELISA employs a biotinylated enzyme inhibitor of Factor VIIa and an anti-FVII/FVIIa monoclonal antibody as the capture antibody. Diluted plasma samples or supernatants containing FVIIa are incubated with the biotinylated inhibitor, which covalently attaches to the FVIIa but not to FVII. The samples are added to microwells precoated with the FVIIa capture antibody. FVIIa is detected by binding of the streptavidin conjugated horseradish peroxidase (HRP) conjugate to the immunocaptured FVIIa/biotinylated inhibitor complex. The addition of TMB substrate and its subsequent reaction with HRP provides a blue color. Sensitivity is increased by addition of a 0.5N sulfuric acid stop solution, yielding a yellow color. FVIIa levels are determined by measuring sample solution absorbance at 450 nm and comparison against those of a standard curve developed using known amounts of fVIIa.

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