The development of immunoassays for the detection of bovine brucellosis and aflatoxin B1

The research discussed in this thesis focuses on the development and characterisation of immunoassays for the detection of aflatoxin Bi (AFBi), a toxic fungal metabolite, and for the diagnosis of bovine brucellosis, a highly contagious disease of cattle caused by B ru c e lla abortus. An AFBi-specific single chain fragment variable (scFv) was isolated from a preimmunised phage display library and the gene encoding it sub-cloned into a range of different vectors for soluble expression o f monomeric, dimeric and bifunctional scFvs. The genetically-derived scFvs were then applied to the development of competitive ELIS As and BIAcore-based inhibition assays for the detection o f AFBi. A lateral flow immunoassay (LFIA) was developed for the detection of AFBi using an AFBi-specific monoclonal antibody. Each immunoassay format described was suitable for the detection of AFBi, with high levels of sensitivity, specificity and reproducibility achieved. Several immunoassay formats for the diagnosis of bovine brucellosis, in serum samples, were investigated. Four antigens were selected as diagnostic markers for brucellosis and included whole B. abortus cells, a crude cytoplasmic lysate, an 18kDa cytoplasmic protein ( p i8) and a 26kDa periplasmic protein (bp26). Recombinant forms of the p i 8 and bp26 proteins were cloned and expressed using a high-level expression vector in E.coli. Two polyclonal antibodies, directed against whole B.abortus cells and the crude cytoplasmic lysate, were developed and a naive phage display library was used to isolate scFvs directed against the recombinant bp26. Feasibility studies were carried out on the indirect ELISAs incorporating the four antigens and on the sandwich ELISAs with the polyclonal antibodies. The indirect and sandwich ELISAs, for the diagnosis of bovine brucellosis, were then validated using a panel of Brucella - positive and negative serum samples