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A fluid phase immunoradiometric assay (IRMA) specific for factor VIII coagulant antigen determinants (VIII:CAg) employing a human Fab’ fraction antibody has been used to assay purified bovine factor VIII. Bovine plasma, although 8-10 times as active in the one-stage clotting assay for VIII:C is only 40% as relative to human plasma in the in VIII:CAg and exhibits a parallel dose response curve. Thrombin (0.1 units/ml) enhances purified bovine VIII:C 20-fold or more but shows no effect on VIII:CAgΛ higher thrombin concentration (30 units/ml) destroys 97% of the VIII:C activity but only about 30% of the VIII:CAg. Similarly, both human and bovine serum retain about 70% of the VIII:CAg relative to the respective plasma levels. Trypsin (5 ug/ml1 hr 1 destroyed over 99.5% of the factor VIII:C in a purified bovine factor VIII preparation., but left a similarly high (70-90%) level of VIIl:CAg. EDTA-aged plasma used as an artificial substrate for factor VIII:C contains an undiminished level of VIII:CAg, but heated plasma (56°, 1 hr.) showed parallel losses (over 95%) of both VIII:C and VIII:CAg. The stability of factor VIII:CAg to protease treatment should permit characterization of purified factor VIII in caaes where biological activity is absent |
