Characterization of antibodies in patient plasma.
(A) Binding of antibodies to radiolabeled recombinant factor VIII fragments corresponding to the A2 domain (A2) and an A2 domain in which residues Arg484–Ile508 were replaced for the corresponding sequence of factor V (A2-FV484-508) was assessed by immunoprecipitation analysis. Lane 1, positive control (CLB–CAg 9); lane 2, negative control (normal plasma); lane 3, plasma of patient AMC-67. Molecular weight markers (in kd) are indicated at the right. (B) Neutralization of factor VIII inhibitory activity by recombinant factor VIII fragments. Inhibitor plasma was diluted to a final concentration of 2 BU/mL, corresponding to 25% of residual factor VIII activity. Samples were incubated for 2 hours at 37°C in the presence of increasing concentrations A2 domain (•) and A2-FV484-508 (□). After incubation for 1 additional hour at 37°C in the presence of normal plasma, residual factor VIII activity was determined relative to a sample that was incubated in the absence of an inhibitor.
