MONOCLONAL-ANTIBODIES TO PORCINE FACTOR-VIII COAGULANT AND THEIR USE IN THE ISOLATION OF ACTIVE COAGULANT PROTEIN

Abstract

Partially purified preparations of porcine factor VIII:C were used to immunize mice, and spleen cells from the immunized animals were fused to NS-1 mouse myeloma cells. The ability of hybrid culture fluids to bind factor VIII:C was detected with a radiolabeled, affinity-purified, human antihuman VIII:C inhibitor. Three cloned hybrid lines were obtained that preferentially bind to VIII:C when compared to von Willebrand factor binding. Two of these monoclonal antibodies partially inhibit VIII:C coagulant activity. The 3rd antibody does not inhibit VIII:C but it can be used as an affinity reagent to absorb dissociated VIII:C out of solution. Active coagulant can be recovered by elution in 50% ethylene glycol. The VIII:C obtained has a specific activity of 6 units/.mu.g based on absorbance measurements. When analyzed on SDS [sodium dodecyl sulfate] gels, the unactivated VIII:C contains 3 bands of apparent MW 166,000, 130,000 and 76,000. Thrombin treatment results in a 40-fold increase in activity and cleavage to products of 76,000, 67,000 and 50,000 and small amounts of lower MW peptides. EDTA inactivation of the factor VIII:C results in the separation of the 166,000 and 130,000 chains from the 76,000 chain, suggesting a Ca2+ dependent noncovalent interaction among the chains.