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Chemical modification and immunological studies on recombinant factor VIII

Manning, Fiona (1994) Chemical modification and immunological studies on recombinant factor VIII. PhD thesis, Dublin City University.

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.
Item Type:Thesis (PhD)
Date of Award:1994
Supervisor(s):O'Kennedy, Richard and Fagan, Ciarán
Uncontrolled Keywords:Blood Coagulation; Blood disorders
Subjects:Biological Sciences > Biotechnology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
ID Code:18983
Deposited On:28 Aug 2013 10:03 by Celine Campbell . Last Modified 28 Aug 2013 10:03

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