The production of polyclonal, monoclonal and genetically-derived scFv antibody fragments for the detection of the ß-lactam antibiotic, cephalexin, in milk

Abstract

This thesis describes the production of antibodies for the detection of cephalexin and their application in ELISA and Biacore ‘real-time’ surface plasmon resonance (SPR)-based biosensor assays. The use of antibiotics in food-producing animals has many beneficial effects (e.g. treatment of mastitis in dairy cattle). However, their use and misuse has also been implicated in the increased incidence of resistant microbial strains with the associated public health risks and economic losses for food processors. Antibiotic residues can adversely affect bacterial starter cultures used in the production of milk-based products such as cheese and yoghurts. The P-lactam antibiotic cephalexin was chosen as the target for the production of antibodies due to its generic cephem-based structure, solubility, and, in particular its use of this antibiotic in treatment of udder infections (e.g. mastitis). Cephalexin-protein conjugates were produced and used for the generation of polyclonal and monoclonal antibodies that were then purified and characterised by SDS-PAGE, Western blotting and ELISA. The specificity of these antibodies was determined and they were used in displacement immunoassays for the detection of the relevant antibiotic in ‘spiked’ samples of PBS and whole milk. Recombinant single chain Fv (scFv) antibody fragments were produced using phage-display technology. Naive and immune phage-display libraries were panned using cephalexin conjugates. In addition, a monoclonal antibody-secreting hybridoma cell line was used in the production of a phage-displayed recombinant scFv. The recombinant scFv (wild type) derived from the parental hybridoma cell was purified and characterised by SDS-PAGE, Western blotting and ELISA. Wild type scFv was subsequently used as a template for the production of a mutant scFv phage-displayed library using random mutagenesis and error-prone PCR.Mutant scFv antibodies were isolated, expressed and then purified using immobilised metal affinity chromatography (IMAC). Two mutant clones expressing scFv antibodies selected showed improved functional expression and assay performance when compared to wild type scFv. Comparative gene and amino-acid sequence analysis and protein modelling was carried out on wild type and selected mutant recombinant scFv antibodies and confirmed the presence of mutations in the framework and variable heavy chain complemetarity determining region amino acid sequences. All antibodies were evaluated in ELISA and Biacore assay formats for the detection of cephalexin in milk samples. The polyclonal, monoclonal and recombinant scFv antibodybased assays demonstrated varying sensitivity and reproducibility but achieved cephalexin detection below the legislated European Union maximum residue limit (EUMRL) in milk ( 10 0 Mgflig)