MEASUREMENT OF ANTI-FACTOR-VIII IGG, IGG4 AND IGM ALLOANTIBODIES IN PREVIOUSLY UNTREATED HEMOPHILIA-A PATIENTS TREATED WITH RECOMBINANT FACTOR-VIII

Using enzyme-linked immunosorbant assay (ELISA), we measured anti-fact or VIII immunoglobulin G (IgG), IgG4 and IgM in serum samples obtained from hemophilia A patients treated with a recombinant factor VIII (rF VIII) preparation (Kogenate(R)), Twelve pre- and post-treatment serum samples from 3 previously untreated patients who developed an inhibito r (alloantibody) were evaluated. Both rFVIII and plasma-derived factor VIII (pdFVIII) were used as antigen. Serum samples were diluted 37.5- fold. For the measurement of IgM antibody, protein A Sephadex suspensi on was added to the patients' serum samples and the supernatant was as sayed. Anti-human IgG, IgG4, and IgM monoclonal antibodies labelled wi th peroxidase were added and absorbance at 450nm was determined, The r eactivity of the IgG antibody with rFVIII and pdFVIII was extremely lo w. Samples containing the IgG4 inhibitor with a neutralizing activity of approximately 7.5 Bethesda units (BU)/ml resulted in absorbance val ues of 0.451-0.551, thus demonstrating a considerably high degree of s ensitivity. The correlation between the neutralizing activity and reac tions of the IgG4 antibody with rFVIII and pdFVIII antigens was high, with correlation coefficients of 0.912 and 0.966, respectively. Furthe rmore, the correlation coefficient between the measured absorbance val ues for the antibody reacted with pdFVIII and rFVIII was 0.961. No cor relation was found between the reactivity of IgM to rFVIII and pdFVIII .