Production and application of monoclonal antibodies suitable for the specific detection of listeria monocytogenes
Hearty, Stephen (2005) Production and application of monoclonal antibodies suitable for the specific detection of listeria monocytogenes. PhD thesis, Dublin City University.
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The principal objective of this research concerned the production, characterisation and application of antibodies for the specific detection of Listeria monocytogenes.
Monoclonal antibodies were generated against various L. monocytogenes-derived antigens. Initially, a panel o f antibodies was produced against a 60 kDa extracellular protein isolated from exponentially growing L. monocytogenes culture supernatants.Subsequently, the L. monocytogenes-speclfic surface pathogenicity protein, Intemalin B (InlB), was biochemically isolated and used to generate InlB-specific monoclonal antibodies. The specific reactivity of these antibodies was demonstrated using purified recombinant InlB (rlnlB) and p60 (rp60) proteins, respectively. However, the anti-p60 antibody was cross-reactive with certain non-pathogenic members of the Listeria genus and the anti-InlB monoclonal antibody was not capable of efficiently binding InlB on the L.monocytogenes cell surface.
Thus, a third panel of monoclonal antibodies was generated using intact, formalininactivated L. monocytogenes cells as the immunogen. From this panel, a promising antibody was selected and shown to demonstrate improved specificity for the L.monocytogenes species. This antibody was further examined in order to demonstrate its potential application in traditional immunoassay and SPR-based biosensor formats.
The L. monocytogenes inlA gene was cloned into a compatible expression vector and recombinant InlA protein (rlnlA) was heterologously expressed in Escherischia coli. This was then used to confirm that the antibody was indeed specific for the L. monocytogenes pathogenicity marker, InlA. The results indicated that the anti-InlA monoclonal antibody produced was capable o f specifically binding to intact L. monocytogenes cells and therefore, it was concluded that it could potentially be employed to facilitate more rapid, reliable and specific detection o f L. monocytogenes cells.
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