Immunoaffinity purification of factor VIII complex.
Murine monoclonal antibodies to human von Willebrand factor (vWf) were immobilised on Sephacryl S-1000. Various solutes were screened for their ability to elute 125I-vWf from the immobilised antibodies. The most effective solutions were then tested to determine which allowed retention of factor VIII procoagulant activity (VIII:C) and activity of vWf measured by platelet aggregation in the presence of ristocetin (Ristocetin cofactor activity R. cof.). Finally, F VIII complex was purified from both plasma and cryoprecipitate by immunoaffinity chromatography under the selected conditions. The product had a specific activity of 45 units of VIII:C per mg of protein and 60 units of R. cof. per mg representing a 4000-fold purification from plasma. The fibrinogen and fibronectin content were each less than 4% of the total protein with vWf accounting for 60% of the total protein in the final product. Multimer analysis of the product showed a similar pattern to normal plasma and contamination by murine monoclonal antibody was less than 300 ng per mg of protein. A novel product is thus obtained containing both clinically relevant VIII:C and R. cof. in a single vial whilst using only one specific monoclonal antibody.
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D000941:Antigens / Q000302:isolation & purification; D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007163:Immunosorbent Techniques; D012310:Ristocetin / Q000502:physiology; D014841:von Willebrand Factor / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000941:Antigens; D002021:Buffers; D014841:von Willebrand Factor; D012310:Ristocetin; D005169:Factor VIII