Isolation of Specific Homologous Antibodies to Factor VIII by Immunoadsorption

Current immunological studies of Factor VIII use heterologous antibodies which predominantly measure Willebrand Factor (WF) and give little information on Factor VIII procoagulant activity (VIII:C). Purification of homologous antibodies specific for VIII:C has been hampered by the fact that they do not form immune precipitates. We have attempted to isolate such antibodies by solid phase immunoadsorption and subsequent elution. Human Factor VIII was specifically bound to goat anti-human Factor VIII IgG previously immobilized onto Sepharose 6 B beads. IgG isolated from a Haemophiliac with a high titer anti-VIII:C antibody (700 Oxford U/ml) was labeled with 125-I and reacted for 72 hours with these beads. The column was then washed with 0.1 M glycine, 0.5 M NaCl, pH 10 buffer to remove non specifically adsorbed material. Specifically adsorbed material was then eluted with 2.5 M MgCl2, pH 7.3, and the peak of radioactivity was filtered on Biogel A-5 m. Both anti-VIII:C and radioactivity were recovered in three distinct peaks. The third peak, corresponding to IgG, contained anti-VIII:C activity with a 10 fold purification as estimated by specific radioactivity. The second peak, eluting just in front of the IgG, had half the specific radioactivity of the third peak. The first peak, corresponding to the Vo, contained Factor VIII related antigen and very little anti-VIII:C activity. The pattern by SDS-polyacrylamide gel electrophoresis is compatible with the existence of Factor VIII (WF-VIII:C)-anti-VIII:C complexes in the first peak; VIII:C-anti-VIII:C complexes in the second, and free anti-VIII:C IgG in the third one. Thus the method leads to the formation of stable VIII:C-anti-VIII:C complexes, allowing the purification of specific human anti-VIII:C antibodies.