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Development of a flow cytometric bead immunoassay as an aid to potency evaluation of enterotoxaemia vaccines
Dissertation (MSc)--University of Pretoria, 2012.
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University of Pretoria
2013
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| _version_ | 1867613468200271872 |
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| access_status_str | Open Access |
| author2 | Theron, Jacques |
| author_browse | Theron, Jacques |
| author_facet | Theron, Jacques |
| collection | Thesis |
| dc_rights_str_mv | © 2012 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria E13/4/445/g |
| description | Dissertation (MSc)--University of Pretoria, 2012. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/30924 |
| institution | University of Pretoria (South Africa) |
| last_indexed | 2026-06-10T12:36:37.472Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2013 |
| publishDateRange | 2013 |
| publishDateSort | 2013 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/30924 Development of a flow cytometric bead immunoassay as an aid to potency evaluation of enterotoxaemia vaccines Theron, Jacques Crafford, Jan Ernst Buys, Angela UCTD Dissertation (MSc)--University of Pretoria, 2012. Enterotoxaemia is an economically important disease of sheep, goats and calves. The disease affects mainly young animals aged between four to six months. Enterotoxaemia is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens Type D. Due to almost certain death of affected animals, there is no form of treatment. The only practical means of controlling the occurrence of enterotoxaemia is to immunize animals through vaccination. The vaccine is prepared by toxoiding the bacterial culture filtrate, and contains a range of proteins in addition to the epsilon toxin. Batches of the vaccine are thus required to be tested for safety, efficacy and potency. The potency of the vaccines is currently tested with the in vivo mouse neutralisation test (MNT). However, due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study, an indirect cytometric bead immunoassay (I-CBA) was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect ELISA (I-ELISA) and the MNT. To investigate, three groups of eight guinea pigs were immunized with one of three different production batches of the enterotoxaemia vaccine. Guinea pig sera were collected prior to vaccination and at five weeks post-vaccination, and the sera of four guinea pigs per group were pooled to give six test sera. The levels of anti-epsilon toxin antibodies in the respective test sera were subsequently determined using MNT, I-ELISA and I-CBA. The I-CBA assay developed during the course of this study is based on coating of functional beads with purified epsilon toxin that serves to capture anti-epsilon toxin antibodies from the test sera. Following incubation with fluorescein isothiocyanate (FITC)-labelled anti-guinea pig IgG, the samples were analyzed by flow cytometry and the anti-epsilon toxin antibody concentrations were extrapolated from a standard curve using linear curve fitting. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels of the guinea pig test sera determined by both the ICBA and I-ELISA tests were higher than that of the MNT assay which is accepted as the current “gold standard”. Moreover, in contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (i.e. not less than 5 U/ml). These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimization and standardization of the I-CBA assay described in this study. Microbiology and Plant Pathology MSc Unrestricted 2013-09-09T07:51:45Z 2013-06-28 2013-09-09T07:51:45Z 2013-04-12 2012 2013-06-20 Dissertation Buys, A 2012, Development of a flow cytometric bead immunoassay as an aid to potency evaluation of enterotoxaemia vaccines, MSc Dissertation, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/30924> E13/4/445/gm/ http://hdl.handle.net/2263/30924 http://upetd.up.ac.za/thesis/available/etd-06202013-142040/ © 2012 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria E13/4/445/g application/pdf University of Pretoria |
| spellingShingle | UCTD Development of a flow cytometric bead immunoassay as an aid to potency evaluation of enterotoxaemia vaccines |
| title | Development of a flow cytometric bead immunoassay as an aid to potency evaluation of enterotoxaemia vaccines |
| title_full | Development of a flow cytometric bead immunoassay as an aid to potency evaluation of enterotoxaemia vaccines |
| title_fullStr | Development of a flow cytometric bead immunoassay as an aid to potency evaluation of enterotoxaemia vaccines |
| title_full_unstemmed | Development of a flow cytometric bead immunoassay as an aid to potency evaluation of enterotoxaemia vaccines |
| title_short | Development of a flow cytometric bead immunoassay as an aid to potency evaluation of enterotoxaemia vaccines |
| title_sort | development of a flow cytometric bead immunoassay as an aid to potency evaluation of enterotoxaemia vaccines |
| topic | UCTD |
| url | http://hdl.handle.net/2263/30924 http://upetd.up.ac.za/thesis/available/etd-06202013-142040/ |