Solid-Phase Immunoradiometric Assay of Factor-VIII Protein

A solid phase immunoradiometric assay for factor-VIII protein has been developed using ¹²⁵I-labelled rabbit antibody made against highly purified factor VIII. Antibody specific for high molecular weight factor-VIII protein was isolated using an immunoadsorbent consisting of highly purified factor VIII bound to diazotized m-aminobenzyl (oxymethyl)-cellulose. The purified anti-factor VIII antibody was labelled with ¹²⁵I while bound to the immunoadsorbent and then eluted at pH 2.9. Dilutions of plasma samples for assay were incubated for 48 h in anti-factor VIII antibody-coated tubes. The tubes were washed to remove unbound proteins and the ¹²⁵I-labelled, purified antibody was added. After 48 h incubation the tubes were washed to remove unbound antibody and counted. The concentration of immunoreactive factor-VIII protein in pooled normal human plasma was determined to be 8 μg/ml. The minimum amount of factor VIII measured by the assay is less than 0.16 ng. Factor-VIII protein was present in normal concentration in haemophilia A plasma, and in reduced concentration in von Willebrand's disease plasma. This method has the advantages of improved sensitivity, specificity, efficiency and economy as compared with previous factor-VIII immunoassays.