| Popis: |
New methods for the determination of human factor VIII clotting protein (F. VIII:C) have been developed utilizing hemagglutination technique without using any other coagulation proteins in the assay. A 50% ammonium sulphate precipitate of the plasma from a hemophilic patient containing high titer of F. VIII:C inhibitor was purified on an affinity column of sepharose 4B covalently linked to F.VIII:C by CNBr. The 2.8 M MgCl2 eluted protein, after dialysis against 0.1 M phosphate buffered saline (pH 7.4), was coupled on either fresh or stabilized human erythrocytes by using either chromic acid or glutaraldehyde as the coupling agent. Serial dilutions of human plasma or F. VIII concentrate were added to the anti-F.VIII:C coated red cell suspension on a microtiter plate. For the stabilized cells, the plate was incubated on a vibration-free surface at room temperature. The results were read after two hours. For the fresh cells, the plate was incubated at room temperature for 30 minutes and was centrifuged at 300 Xg for ½ minute. It was then placed on a tilted platform at a 600 angle. The results were read after 10 minutes. By these methods, it is possible to determine the detectable level of F.VIII:C in plasma and the F.VIII:C concentration in cryoprecipitates and F.VIII concentrates. |
