FVIII antibodies can be detected using indirect enzyme-linked immunosorbent assay (ELISA). FVIII antibodies in patient sera, whether inhibitory or 'immune', are captured in microtitre plates coated with recombinant FVIII. Unbound material is then washed off and a solution of antibody to human immunoglobulin that is conjugated to an enzyme is added to ‘tag’ onto any captured FVIII antibodies. Unbound conjugate is washed off and a substrate for the enzyme is added, the product of the enzyme-substrate reaction being coloured. Colour intensity is in direct proportion to the degree of conjugate-binding, itself proportional to the amount of FVIII antibody capture. The assay is not quantitative and is reported as either positive or negative based on whether the colour intensity exceeds a kit-specific cut-off value.
Acquired inhibitors to FVIII are antibodies that arise in 10-15% of patients with hereditary haemophilia A as a result of recognising therapeutic FVIII products as 'foreign' protein, or in patients with previously normal haemostasis as part of an autoimmune process. Inhibitors in congenital haemophiliacs complicate treatment as the infused FVIII has a shorter survival time, and patients with acquired haemophilia can present with severe bleeding. Some patients generate antibodies that are not inhibitory but may cause increased clearance upon removal of immune complexes from the circulation.