Comparative measurement of anti-factor VIII antibody by Bethesda assay and ELISA reveals restricted isotype profile and epitope specificity.

Factor VIII (FVIII) inhibitor antibodies are produced against functional epitopes of FVIII in about 30% of severe hemophilia A patients leading to inhibition of its procoagulant activity. The Bethesda assay, the most commonly used method to measure FVIII inhibitors, based on inhibition of coagulant activity of FVIII, is neither able to detect noninhibitory antibodies nor their isotype. In this study we employed an indirect enzyme-linked immunosorbent assay (ELISA) to measure dif ferent isotypes and IgG subclasses of anti-FVIII anti body in the plasma of hemophiliacs (with and without inhibitor) and normal individuals using recombinant FVIII-coated microtiter plates. The results showed a predominance of IgG and IgG4, though IgA was slightly elevated in a few inhibitor-positive patients and IgM was hardly detectable. A highly significant correlation was found between the Bethesda titer and the optical density values of total Ig, IgG and IgG4 anti-FVIII antibodies obtained by ELISA (p<0.0001). These findings suggest a restricted specificity of anti-FVIII response in hemophiliacs towards functional epitopes of the molecule. Furthermore, high specificity and reasonable sensitivity of the ELISA, together with other technical advantages, suggest this method as a suitable supplementary technique for rapid large-scale screening of inhibitor-positive samples, though ELISA-negative samples need to be rechecked by the Bethesda assay to identify patients with a low inhibitor titer.