Production of antibodies for use in a biosensor-based assay for Listeria monocytogenes

The inclusion of L. monocytogenes in the list of organisms subject to HACCP has recently driven the search for detection methods suitable for on-line monitoring. The aim of the work presented in this thesis was the development of a biosensor-based immunoassay for the detection of Listeria monocytogenes using SPR. Two polyclonal antibodies were generated from Listeria monocytogenes MB extract and heat-treated Listeria monocytogenes cells. Both antibodies were purified and characterised by ELISA, SDS-PAGE and Western blotting. An inhibition ELISA-based immunoassay was developed with each antibody for the detection of Listeria monocytogenes. Intra- and interday studies were performed to evaluate the accuracy and intermediate precision of the assays. The feasibility of detecting Listeria monocytogenes cells in chocolate milk, a food matrix which has been reported to have been the cause of a well known Listeria monocytogenes-&ssoc\atQ<\ food poisoning outbreak, was also examined. To determine the potential cross reactivity of each antibody, inhibition ELISAs were performed with a number of bacterial strains. It was concluded that both antibodies can be used as valuable tools for the genus-specific detection of Listeria cells, but were severely limited for the species-specific detection of Listeria monocytogenes cells. Expressing Listeria monocytogenes invasion-associated proteins in E. coli allows the safe and efficient production of high quantities of pure protein for use in the generation of Listeria monocytogenes specific antibodies and immunoassay development. Two Listeria monocytogenes invasion-associated proteins, Intemalin B (InlB) and p60 (also known as iap), were cloned and expressed in E. coli XL-10 Gold cells. Expressed protein was purified by immobilised affinity chromatography (IMAC) and used for the selection of specific antibodies (Chapter 5) and for the development a biosensor-based immunoassay for the detection of Listeria monocytogenes (Chapter 6). The emergence of recombinant antibody phage display technology has transformed the way we generate antibodies for the specific detection of a chosen analyte. Chapter 5 describes the development of two combinatorial antibody libraries from mice immunised with Listeria monocytogenes cells and InlB protein extract. Phage scFv antibodies were selected from both murine antibody libraries and from a large naive human antibody library against Listeria monocytogenes cells and invasion associated protein. A number of the selected phage-scFv antibodies could not be expressed as soluble scFv and also showed tendencies to cross react with various bacterial strains tested. A soluble scFv antibody was selcctcd that recognised the Listeria monocytogenes invasion-associated protein, Internalin B, but did not recognise Listeria monocytogenes cells. An inhibition ELISA was developed with this antibody to indircctly detect Listeria monocytogenes. The two polyclonal antibodies and the selected anti-InlB scFv antibody were used in development of three biosensor-based immunoassays using a BIAcore 3000 instrument (chapter 6 ). Various assay formats and sensor chip surfaces were evaluated. Intra- and interday assay variability studies performed to determine the precision and reproducibility of each assay. The assay developed with the polyclonal anti-InlB polyclonal proved to be most sensitive and cost effective while the assay developed with the anti-InlB scFv antibody fragment proved most specific for the detection of Listeria monocytogenes.