Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease that affects goats in Africa and Asia resulting in great economic losses. The aetiological agent is Mycoplasma capricolum subsp. capripneumoniae (Mccp). It belongs to a group of organisms that are all associated with economically important diseases of ruminants and all exhibit similar genetic and antigenic characteristics. The diagnosis of CCPP has often been considered difficult due to confusion with other mycoplasmas of ruminants and the limited specificity of most diagnostic assays. It is therefore important to improve diagnosis and thereby the control of CCPP. Two approaches, namely phage display and anti-idiotype antibodies can be used to identify antigenic mimics with the potential to be used in developing new vaccines, diagnostic methods or therapeutics. Both were used in an attempt to generate surrogate antigens for Mccp. A monoclonal antibody (Mab) or its F( ab ')2 fragments directed against Mccp membrane protein epitopes was injected into hens to induce the production of anti-idiotype antibodies. The antibodies produced were functionally able to mimic the epitope recognised by the Mab since they inhibited binding of the Mab to mycoplasmal lysate. Mice were also immunised with the Mab and or F(ab') 2 fragments of the antibody. The resulting antisera were tested in ELISA, but no significant response was detected. The selection of peptides from a random epitope library displayed on the surface of filamentous phages was used to characterise the epitope recognised by the Mab. Two different, but related peptides were identified that reacted with the antibody in enzyme-linked immunosorbent assays (ELISA). Binding to the Mab was further characterised by surface plasmon resonance. Sequence analysis revealed that the two peptides each had a cysteine residue in addition to the one fixed in amino acid position 2 as well as identical or similar amino acid residues in positions 5 (P), 8 (I/L) and 13 (L). One of the peptides had 74% similarity with an amino acid sequence of the PG 1 strain of Mycoplasma mycoides subsp. mycoides SC. The peptides as well as the anti-idiotype antibodies were not detectable using Mccp-specific goat antiserum suggesting that the serum did not contain paratopes that could accommodate the surrogate epitopes. In spite of this, the antiserum efficiently inhibited the binding of the Mab to immobilised mycoplasmal lysate in a standardised test for Mccp antibodies. This assay therefore appears to depend on a structural, rather than a functional blocking of the epitope which the Mab recognises. The findings in this study have elucidated some of the characteristics of the Mab and raised the possibility that the epitope recognised by the Mab is not immunodominant. Peptides identified by phage display and chicken/murine anti-idiotype antibodies, nevertheless, have the potential of being used as antigens in immunoassays aimed at CCPP diagnosis. In light of the results generated it would therefore be necessary to investigate other Mabs or polyclonal antiserum in order to yield antibodies or peptides of the desired specificity. / Dissertation (MSc (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/23600 |
| Date | 30 March 2006 |
| Creators | Benguric, Dubravka Roka |
| Contributors | Prof L H Nel, upetd@up.ac.za, Dr D H du Plessis |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2000, 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. |
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