SummaryHuman factor VIII (f VIII) inhibitors are pathologic antibodies that inactivate fVIII. A cDNA clone was modified to encode f VIII amino acid residues 373-740 for expression in a baculovirus vector in insect cells. The encoded protein fragment H2 was produced as a soluble, secreted protein, and it was used to test inhibitor plasmas for the presence of antibodies that were not detected by immunoblotting. Seven of 13 inhibitors that bound, only to the fVIII light chain by immunoblotting also bound to fragment H2 in an immunoprecipitation assay. Thus multi-chain inhibitor reactivity of inhibitors is more frequent than previously reported. One of these inhibitors was shown to share the epitope for other inhibitors that bind to H2 within amino acid residues 373-541 in immunoblotting assays. The sensitive immunoprecipitation assay described allows determination of relative H2 binding capacity of the total IgG and epitope localization of inhibitors that cannot be similarly characterized by immunoblotting.
Summary. Human erythrocyte ghosts where prepared by osmotic lysis and washed thoroughly with deionized distilled water. The resultant stroma were extracted twice with n‐butanol producing 81.8 % ± 2.7 solubilization of the membrane protein. The initial extract contained 5 % of the lipid present in intact ghosts. The second extract contained no detectable lipids. The solubilized glycoprotein possessed A, B and H blood group activity comparable to that of the intact ghosts at the same protein concentration. Fractionation studies suggested that the maximum serological activity was associated with a high molecular weight structure.
In contrast to most previous work demonstrating A, B and H blood group activities to be exhibited by erythrocyte glycolipids this study reports these blood group specificities to be present in human erythrocyte membrane glycoprotein.
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