Factor VIII (aka Factor VIII:C or Antihemophilic Globulin) is a glycoprotein zymogen that circulates in a stabilized non-covalent complex with von Willebrand Factor (vWF). Following activation by thrombin or Factor Xa, Factor VIIIa dissociates from vWF and catalyzes the activation of Factor X by Factor IXa in the amplification phase of coagulation. Factor VIIIa activity is quickly decreased by spontaneous dissociation and proteolytic degradation by activated Protein C, Factor Xa and Factor IXa. Hemophilia A is caused by mutations in the Factor VIII gene; a majority of patients have decreased Factor VIII plasma levels while 5% of patients have normal levels of nonfunctioning protein. The sensitive quantitative measurement of total human Factor VIII antigen in plasma samples is easily performed with this 96 well strip format ELISA kit. The average normal plasma level of Factor VIII is defined as 1.0 IU/ml and the normal range is 0.4-1.8 IU/ml. Hemaophilia A patients are classified by the following Factor VIII levels: 0.05-0.25 IU/ml = mild, 0.01-0.05 IU/ml = moderate, and <0.01 IU/ml = severe. The assay measures total human Factor VIII in the 0.0016-0.84 IU/ml range. Samples giving human Factor VIII levels above 0.84 IU/ml should be diluted in blocking buffer before use. 1:8 and 1:16 dilutions for normal plasma, or 1:4 and 1:8 dilutions for Haemophiliac plasma, are suggested for best results.

Human Factor VIII will bind to the affinity purified capture antibody coated on the microtiter plate. After appropriate washing steps, biotin labeled anti-human Factor VIII primary antibody binds to the captured protein. Excess primary antibody is washed away and bound antibody is reacted with peroxidase conjugated streptavidin. Following an additional washing step, TMB substrate is used for color development at 450nm. A standard calibration curve is prepared along with the samples to be measured using dilutions of human plasma. Color development is proportional to the concentration of Factor VIII in the samples. All reagents and standards are provided in these ELISA kits.

The Factor VIII level in the human plasma standard provided is calibrated against a secondary standard that is referenced to the WHO or ISTH International Standard.