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

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