| Summary: | Factor VIII is a complex of two proteins, the von Willebrand factor (or factor VIII related antigen) and the procoagulant protein. Both are essential for normal haemostasis. Problems exist in the purification of factor VIII as it is present in only low concentrations in plasma, and its procoagulant activity is unstable. As a result, therapeutic factor VIII concentrates prepared by the Blood Transfusion Service (for the treatment of heamophilia and von Willebrand's disease) are relatively impure, and the yield from existing purification processes is low. The studies presented in this thesis are aimed towards increasing the quality and yield of therapeutic concentrates. Attention has been focussed on improving methods for the purification and assay of factor VIII. Novel affinity purifications reagents for factor VIII have been studied, and methods for removing the major impurity (fibrinogen) from conventional factor VIII concentrates have been investigated. The factor VIII related antigen (FVIIIR:Ag) and the procoagulant antigen (FVIII:CAg) have been purified, and used as immunogens for the production of specific antibodies. A large volume of polyclonal antibody to FVIIIR:Ag has been produced in sheep. This was subsequently used to develop an immunopurification method for FVIII:CAg. Immunisation of mice with purified FVIII:CAg gave a valuable panel of ten monoclonal antibodies to procoagulant factor VIII. These have important applications in the assay, purification and biochemical study of this protein. Sensitive radiometric assays for FVIIIR:Ag and FVIII:CAg have been established. This work involved the development of methods for the preparation of ¹²⁵I-FVIIIR:Ag, and for the purification and labelling of human anti-FVIII:CAg Fab' fragments from inhibitor plasma. An artificial factor VIII-deficient substrate has been prepared on a large scale for the one-stage bioassay of procoagulant activity (FVIII:C).
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