Chemical modification and immunological studies on recombinant factor VIII

Factor VIII (FVIII) is a high molecular weight glycoprotein which is deficient or functionally defective in Haemophilia A. It circulates in normal plasma as part of a complex with von Willebrand factor (vWF). In the absence of vWF, FVIII is highly unstable. The effects of chemical modifiers on recombinant FVIII (rFVIII) were investigated. A variety of homobifunctional and heterobifunctional chemical cross-linkers and proteinmodifying agents with different side chain specifities was employed. The amino-specific reagents, with the exception of 2-Iminothiolane, caused significant loss of FVIII activity. However, reaction with thiol-specific compounds did not lead to any loss of FVIII activity. It would appear therefore, that amino groups are involved in FVIII activity. Monoclonal and polyclonal antibodies were produced to rFVIII. The effect of binding of these antibodies on FVIII procoagulant activity was investigated. It was found that the binding of the monoclonal antibodies caused loss of activity, whereas, binding of the polyclonal antibodies did not neutralise procoagulant activity. The antibodies were used in the development of enzyme-linked immunosorbent assays (ELISAs) for the detection and quantitation of rFVIII and anti-rFVIII antibodies using a number of different formats. The antibodies were also utilised in the development of a biosensor. Using this device, FVin in normal human plasma and calibration plasma, as well as anti-rFVIII antibodies could be detected. The biosensor also provides a novel method to monitor antibodyantigen interactions. rFVIII was immobilised onto an electrode surface and incubated in a horseradish peroxidase-labe led anti-rFVIII antibody solution. The electrochemical response was based on the enzymic reduction of H20 2 in the presence of an electron mediator (hydroquinone). The oxidised quinone produced was reduced at the electrode surface and current measured