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Title: Studies on parapoxvirus antigens through the development of monoclonal antibodies to orf virus
Author: Housawi, Fadhel Mohammed Taher
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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Twenty-five monoclonal antibodies (mabs) against orf virus, a parapoxvirus (ppv), were produced following the immunisation of mice with a lysate of cells infected with orf-11 virus. These mabs, together with 2 others recloned from an earlier fusion, were identified by ELISA and IFT and characterised. No neutralising activity was shown by any of the mabs. The size of orf proteins detected by the mabs was measured using western blotting and radioimmunoprecipitation (RIP). Western blotting was conducted with two types of orf-11 antigen preparations - gradient purified virus or a lysate of orf-11 infected cells. Five mabs detected a protein of approximately 40 kDa with both purified virus and infected lysate antigens. These mabs detected a protein approximately 65 kDa in size, but only with infected cell lysate antigen. In RIP studies, 21 mabs produced bands 13 of which were against the 65 kDa protein, 7 against that 40 kDa protein while one was against a 50 kDa protein. Twenty-one of the 27 mabs reacted with at least two of 18 vaccinia virus orf virus (VVOV) recombinants expressing a library of orf genome fragments of the NZ-2 virus strain. Four of the mabs which had identified the native 40kDa protein reacted with 2 overlapping recombinants (245 and 247). Seventeen of the mabs, 16 of which had identified the native 65 kDa protein recognised three recombinants 79, 285 and 286 all of which contain different inserts from the same region of the orf virus genome. Subsequent sequencing of the overlapping site between recombinants 245 and 247 by New Zealand collaborators has identified a new orf gene, designated FIL which has been shown to be analogous to the H3L vaccinia virus gene which codes for an immunodominant 35 kDa envelope protein. Cells infected with a new VVOV recombinant expressing only the FIL orf gene showed positive fluorescence with 3 or the 4 mabs which reacted with the 245 and 246 recombinants, confirming the target of these mabs is the product of the FIL gene.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available