Investigations on bluetongue virus using monoclonal antibodies
The main aim of the thesis was the production and characterisation of monoclonal antibodies (Mabs) against bluetongue virus (BTV) and the application of such antibodies to the improvement of BTV diagnosis. The suitability of the indirect ELISA for the detection of antibodies to BTV was examined along with the published protocols for the purification of BTV ELISA antigen. When sera were examined from animals which had experienced either BTV vaccine or any other tissue-culture derived vaccine, host-cell protein contaminants in the BTV ELISA antigen reacted with anti-BHK cell antibodies to give false positive results. None of the published protocols completely removed host-cell protein contaminants. As a solution to these problems monoclonal antibodies were produced to BTV and characterised as to their reactivity against a panel of BTV antigen preparations. They were further characterised by competitive binding assays, and SDS-PAGE analysis of the various BTV preparations with which they reacted optimally. One Mab (3-17-A3), directed against BTV p7, proved suitable for use in a blocking ELISA for the detection of antibodies to all 22 serotypes of BTV. The blocking ELISA proved both more sensitive and more specific than the currently used agar gel precipitin test, and has been adopted as the screening test before importation of semen and embryos into the United Kingdom. A further group of antibodies (characterised by Mab F58) directed against BTV polypeptide NS1, used in a blocking assay detected anti-BTV tubule antibodies. This allows detection of BTV replication in infected animals, and may be used to discriminate between BTV infected animals and animals vaccinated with inactivated BTV vaccine. This work demonstrated the previously unreported BTV-specific nature of BTV NS1. Serum-free suspension culture of the hybridomas and purification of the Mab from tissue-culture supernatant was also developed.