Novel anitbody-based assays for disease and food contaminant detection

This thesis describes the development of immunoassays for the diagnosis of caseous lymphadenitis, a disease of sheep caused by Corynebacterium pseudot~iberculosis; and for the detection of the food contaminants, aflatoxin BI (AFBl), and the p-lactam antibiotic, ampicillin. Imrnunoassays based on the detection of antibodies to a phospholipase D (PLD) exotoxin of Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis (CLA), were investigated. A previously cloned recombinant form of PLD (Menzies et al., 1994) was expressed in E. coli, purified and subsequently used in an indirect ELISA and Biacore assay for the detection of antibodies to C. pseudotliberculosis in sheep serum samples. These assays were optimised using a variety of positive and negative controls and were then applied to a range of clinical samples from Irish herds. A previously isolated Am1-specific single chain variable fragment (scFv) was converted to a chimeric Fab format by the addition of human constant regions, using PCR assembly methods. A mutant Fab fragment was also isolated and demonstrated improved assay performance when compared to the parent scFv. The antibody fragments were successfully applied to the development of several immunoassay formats and compared with regard to sensitivity, specificity, and stability. The scFv fragment was also successfully biotinylated in vivo and applied to the development of an ELISA. Rabbit polyclonal antibodies to ampicillin were produced, characterised and subsequently applied to ELISA and Biacore-based assays for the detection of ampicllin in 'spiked' samples of PBS and whole milk. The antibodies were then employed in the development of a fluorescence-based irnmunoassay for subsequent incorporation into a disposable biochip senor. Naive and immune phage displayed libraries were also screened against a number of ampicillin conjugates for the isolation of an ampicillin-specific scFv