Radioimmunoassay for quantitative measurement of factor VIII-heavy chain

An immunoassay has been developed for the measurement of factor VIII heavy chain (FVIII-HC). IgG from a human inhibitor plasma with specificity for FVIII-HC and FVIII-light chain (FVIII-LC) was coated to microplates with loose wells. After washing, the plates were incubated with sample and after another wash 125I-FVIII-HC was added so that the amount of bound tracer was dependent on the amount of FVIII-HC in sample. When EDTA was included in the assay buffer the assay response was increased 3-fold for normal human plasma. This indicated that the antibody was reactive with a determinant hidden in the FVIII-HC/FVIII-LC complex as EDTA splits the complex. The sensitivity of the assay was 0.004 units/ml (1 unit/ml in normal human plasma pool). Together with a previously published assay for FVIII-LC it has now become possible to measure the relative amounts of FVIII-HC and FVIII-LC in haemophilia A plasma and to study the separate expression of FVIII-HC and FVIII-LC by recombinant DNA technology. Patients with severe haemophilia A had FVIII-HC levels below 0.01 units/ml. However, inhibitor patients in high dose FVIII-treatment showed up to 50 times higher levels of FVIII-HC than FVIII-LC and FVIII:C, indicating the presence of FVIII/anti-FVIII-LC immune complexes. Thus, dependent on assay specificity plasma samples can show very variable content of FVIII:antigen.