An Enzyme-Linked Immunosorbent Assay (ELISA) For Factor VIII Inhibitors

ELISA microtechniques are suitable for measurement of a variety of antigens and antibodies. They are sensitive, safe, and easy to perform. Enzyme-labeled antibodies to factor VIII and factor IX can be used to measure antigen in a “sandwich” assay analogous to the immunoradiometric assay (IRMA). Likewise, specific antibodies can be easily quantitated in plasma samples. Quantitation of F. IX inhibitors by ELISA has been performed by Orstavik and colleagues. We have studied use of the ELISA system for measurement of inhibitors to factor VIII. Purified factor VIII prepared from concentrate is bound to plastic microtiter plates, which are then incubated with plasma dilutions. After washing to remove non-bound material, heterologous anti-human IgG labeled with alkaline phosphatase is added. It binds to any hyman IgG remaining on the plate. Following incubation and washing, the enzyme substrate, p-nitro- phenyl phosphate, is added. Degradation of the substrate produces a color change which is read spectrophoto-metrically. A “through-the-plate” reader allows rapid OD measurements without transfer of the well contents. Ten or more samples in dilution series can be tested simultaneously. The inhibitor titer may be quantitated by comparison with a standard inhibitor measured in Bethesda units by a clotting assay or by direct comparison with the OD curve produced by known quantities of human IgG. Twelve known inhibitors from hemophilia patients and two acquired non-hemophilic inhibitors showed good correlation with the Bethesda unit method. Five normal individuals and five hemophiliacs with no history of inhibitor by clotting assay showed no anti-IX inhibitor by this test.