Visulize fviii antigen kit

FVIII activity was measured by a chromogenic assay using the Coatest SP4 FVIII kit (DiaPharma, West Chester, OH). FVIII antigen was quantified by ELISA using the VisuLize FVIII antigen kit (Affinity Biologicals, Ancaster, ON, Canada). FVIII antigen levels were calculated assuming a concentration of 200 ng/mL in FVIII standard (1 IU/mL). FV antigen was quantified by ELISA using a matched-pair antibody set for human FV antigen from the Affinity Biologicals. FV antigen levels were calculated assuming a concentration of 10 μg/mL in the normal human plasma standard. All assays were performed according to manufacturers’ instructions.

FVIII activity in hemophilia A mouse plasma was determined using an activated partial thromboplastin time assay. Activity was calculated using a standard curve generated from normal human plasma over a range of 0–40% normal FVIII levels. Levels of circulating human Factor VIII protein in mouse plasma samples were determined using the VisuLize FVIII Antigen Kit (Affinity Biologicals, Ancaster, Canada) per manufacturer’s instructions. BDD-FVIII levels were normalized against naive and control vector treated mice to correct for cross reactivity of the ELISA with murine FVIII protein. Inhibitory antibody titers against FVIII were determined using the Bethesda inhibitor assay. One Bethesda unit is defined as the amount of inhibitory antibody that can neutralize 50% FVIII activity following a 2-hour incubation at 37°C. An immunocapture ELISA was used to determine titers of FVIII-specific IgG1 using purified mouse IgG1 as a standard.