Frances Rotblat

Response to infusions of polyelectrolyte fractionated human factor VIII concentrate in human haemophilia A and von Willebrand's disease

Summary. Factor VIII was purified from cryoprecipitate by ion exchange chromatography on solid phase polyelectrolyte E‐5 (PE‐E5). The product was highly purified (3.5 u VIII: C/mg protein) compared to conventional concentrate (0.3 u VIII: C/mg protein) with low fibrinogen, low isoagglutinin titre, and a ratio of factor VIII coagulant activity (VIII: C) to factor VIII related antigen (VIIIR: Ag) of 16:1.

Immunologic studies of factor VIII coagulant activity (VIII:C) 1. Assays based on a haemophilic and an acquired antibody to VIII:C

Abstract A one site immunoradiometric assay for factor VIII coagulant antigen (VIIIC:Ag) is described, using two homologous antibodies to factor VIII, one from a multi transfused haemophiliac and the other from a female with rheumatoid arthritis. Specific C 125 I anti-VIII Fab 1 was prepared from these plasmas and used to set up sensitive (1 and 3 u.dl −1 respectively), rapid (24 hours) assays. Separation of bound from free was obtained by 38% saturated ammonium sulphate precipitation. VIII:C antigen in normal and haemophilic plasmas was assayed using both antibodies. The values were correlated in all samples. There was a small though significant excess of VIIIC:Ag compared to VIII:C. In a small proportion of haemophiliacs VIIIC:Ag was markedly greater than VIII:C. VIIIC:Ag can be detected in the plasmas of patients with acquired inhibitors to factor VIII but not in plasma from haemophilic patients with antibodies. VIIIC:Ag in serum showed a non-parallel dose response curve to plasma, but this could be corrected by the addition of citrated turkey plasma as a carrier. VIIIC:Ag was shown to be more stable than VIII:C at 4°C, 21°C and 37°C. Competitive binding studies carried out by two methods showed that the binding properties of the two antibodies were different. Either the haemophilic antibody can displace the acquired or they are directed against different, although closely related, antigenic sites.

Production of factor VIII deficient plasma by immunodepletion using three monoclonal antibodies

Factor VIII deficient plasma was made from pooled, HIV antibody and hepatitis B antigen screened, normal human plasma by cryoprecipitation and immunodepletion, using three different monoclonal antibodies bound to Sepharose columns, in series. These monoclonal antibodies are specific respectively for von Willebrand factor, factor VIII heavy chain and factor VIII light chain.

Immunologic studies of factor VIII coagulant activity (VIII:C) 2. Factor VIII in selected vertebrates

Cross reactive antigens to factor VIII have been measured in a range of vertebrate phyla. VIII coagulant antigen (VIII:CAg) measured using a human antibody was in general, much lower than reported values of VIII coagulant activity (VIII:C) except in simians and the guinea pig. The dose response curve for the assay was parallel in all cases. Factor VIII-related antigen (VIIIR:Ag) measured using rabbit antibody to human VIIIR:Ag was low or absent in most species except simians and the dose response curve was non-parallel to the human standard except with goat plasma. Avians lacked cross-reactive material.

Affinity Depletion and Affinity Purification of Human Factor IX by Monoclonal Antibodies

Abstract Monoclonal antibodies to human coagulation factors are of potential use in the characterisation, detection and purification of these proteins. In addition, monoclonals could be used to affinity deplete specific factors from plasma to produce deficient substrate for use in one-stage coagulation assays. We have raised four monoclonal antibodies to human factor IX (RFF-IX/1, RFF-IX/2, RFF-IX/3 and RFF-IX/4) and have assessed their suitability for affinity depletion and affinity purification of factor IX. The factor IX deficient plasma so produced is capable of being used as substrate in a one-stage coagulation assay for factor IX and performs as well as does severe Christmas disease plasma. In addition, we have used one of the monoclonal antibodies to obtain highly purified factor IX from factor IX concentrate in a one-step purification procedure.