RABBIT ANTIBODIES AGAINST THE PROCOAGULANT ACTIVITY (VIII-C) OF HUMAN FACTOR-VIII - SOME THEORETICAL AND PRACTICAL CONSIDERATIONS ON THE HUMAN FACTOR-VIII MOLECULE USING HETEROLOGOUS ANTIBODIES

Abstract

The procoagulant activity of factor VIII (VIII:C) was separated from factor VIII related antigen (VIIIR:Ag) by gel filtration in the presence of 0.25 mol/l CaCl2. Antibodies (anti-VIII:C) were obtained by immunization of rabbits with VIII:C. The last step of the purification procedure of antibodies consists of an adsorption on VIIIR:Ag-Sepharose 2 BCL as immunoadsorbent to remove contaminating traces of antibodies against VIIIR:Ag. The anti-VIII:C titer remains unchanged during this adsorption (29 Bethesda U/mg). In solution, anti-VIII:C neutralizes factor VIII activity (in plasma, cryoprecipitate or in purified form) and the fragment VIII:C without reacting with VIIIR:Ag. Once immobilized on a solid matrix (i.e., 2% agarose), it loses over 95% of its inhibitory capacity. The immobilized anti-VIIIR:Ag binds stoichiometrically the antigen and the activity of plasma factor VIII. These results together suggest that factor VIII Is composed of 2 different entities, but undissociated under physiological conditions. Immunophysical analyses as a function of pH and temperature of anti-VIII:C and its complex with factor VIII show properties similar to those of homologous antibodies. The antigen determinants of VIII:C (VIII:CAg) are destroyed at low pH or high temperatures, and VIII:C can no more form a complex with anti-VIII:C. Purified anti-VIII:C was also used in a 2-stage assay to detect VIII:CAg or cross-reacting material in some severe hemophiliacs.