Production and Characterisation of Monoclonal Antibodies to Vibrio cholerae

Ghosh, Souravi (1985) Production and Characterisation of Monoclonal Antibodies to Vibrio cholerae. PhD thesis, University of Glasgow.

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Ogawa and Inaba are the two main serotypes of V. cholerae 01, the etiologic agent of the severe diarrhoeal disease, cholera. In the gut of human hosts, the vibrios produce a cholera toxin which causes rapid dehydration. Various field and volunteer studies (Levine et al, 1979, 1981; Cash et al, 1974) have indicated that antibacterial activity is more important than antitoxin activity in protection against subsequent challenge infection. V. cholerae antigens have been classified by conventional serology into three groups. The A antigen is common to Inaba and Ogawa, the B is specific to Ogawa and the C specific to Inaba. The structural analysis of cholerae LPS has received very limited attention and hence the antigens responsible for homologous and heterologous vibriocidal activity are not well understood. Recent chemical analyses of the cholerae LPS have, however, suggested that the group antigen is a repeat of perosaminyl residues whose amino functions are acylated by L-glycero-tetronic acid (Redmond, 1979; Kenne et al, 1982). However, the type specific antigens of Ogawa and Inaba are still obscure. The objective of the present study was to investigate the shared and unique epitopes of Ogawa and Inaba serotypes with MCAbs. A panel of six MCAbs was produced and assessed for specificity to the two serotypes and other potentially cross reacting bacteria. All were directed primarily to the LPS rather than membrane protein. The specificity profile of each antibody was heavily dependent on the assay system employed and the antibodies could be classified into three groups. Three could be demonstrated to be totally specific to Inaba LPS on some assay systems but to show extensive cross reaction with Ogawa vibrios on other assay systems. Two antibodies were totally specific to Ogawa vibrios on all assay systems. A sixth antibody could be shown to be totally specific on Ogawa vibrios in some assay systems and to be totally specific to Inaba on other assay systems. The dual specificity of the four cross reactive antibodies could be related to the use of washing as opposed to nonwashing assays, the concentration and method of presentation of antigen and antibody, and the density, accessibility and geometry of the antigen on the bacterial surface. These results indicate that the conventional classification of cholerae antigens does not take into account more subtle interactions between antigen and antibody. In addition, they emphasise the point that emerging hybridomas must be screened under the exact conditions of final use of the antibody to be generated. Finally, they indicate that a single antibody may be employed in a dual capacity, detecting different serotypes in two different assay systems. The interaction of the antibodies with the vibrio LPS molecules meant that it was necessary to resolve these molecules in order to analyse the interaction. High resolution electrophoresis was necessary to do this as the size of the repeating unit was discovered to be exceedingly small (less than 300 daltons) compared to that observed for Salmonella typhimurium and coli O111:B4. Most interestingly, no differences in the mobility, number of side chains or the density of the O-chains were detected on SDS-PAGE for the two serotypes. Thus, the difference in serotype cannot be related to difference in electrophoretic mobility even at this fine resolution. Nonetheless, the MCAbs were able to make clear distinctions between the two serotypes on immunoblotting, emphasising their considerable power as a specific detection system. The results suggest that the difference between Ogawa and Inaba must therefore be interpreted in terms of LPS molecules which have 0-side chains with very small repeating units identical in size and electrophoretic mobility but containing enough structural differences to allow them to react differentially with serotype specific monoclonal antibodies.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry, Microbiology
Date of Award: 1985
Depositing User: Enlighten Team
Unique ID: glathesis:1985-76563
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 14:08
Last Modified: 19 Nov 2019 14:08

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