Plasma from two patients with antibodies to factor VIII are being studied to identify the epitopes on factor VIII that are recognized by the antibodies. The general approach has been to enzymatically cleave factor VIII and then immunoprecipitate the resulting fragments using patient immunoglobulin covalently coupled to protein A - Sepharose beads. Following digestion of factor VIII by thrombin, fragments of 50 kD (the A1 region - which is further cleaved to 30 kD and 20 kD pieces), 43 kD (the A2 region), and 73 kD (the light chain consisting of fragments A3, C1, and C2) are generated. Patient B's plasma immunoprecipitates the light chain and both the A1 and A2 fragments, whereas patient P's plasma precipitates the light chain only. As a control a monoclonal antibody with known specificity for an epitope in the aminoterminus of the A2 fragment is used. Immunoprecipitation of trypsin cleaved factor VIII has been unsuccessful. It is unclear as to why, but possible explanations include destruction of the epitopes by digestion and insufficient protein to be detected by immunoblotting.
