Antibody-based biosensor assays for the detection of zilpaterol and markers for prostate cancer

Abstract

The research presented in this thesis describes the production and application of antibodies against the drug of abuse zilpaterol, and the application of antibodies against prostate-specific antigen (PSA), a cancer marker. Polyclonal antibodies were used in the development of immunoassays in a competitive ELISA format and on the Biacore (a surface plasmon resonance-based optical biosensor capable of monitoring biomolecular interactions in 'real-time'). A zilpaterol-HSA conjugate was used to generate and characterise single chain antibody fragments. A combinatorial single chain (scFv) antibody phage display library was generated to zilpaterol. Splenomic mRNA from mice pre-immunised with a zilpaterol-HSA conjugate was used in the amplification of antibody genes followed by cloning into vectors from a well-established phage display system. Four positive clones were isolated during panning. One clone (Bl) was selected and re-cloned into a plasmid fiom soluble scFv antibody expression. The soluble scFv antibody was purified and used in the development of a competitive ELISA-based assay. Further analysis of the B 1 clone was carried out during the development of an inhibition assay for zilpaterol on Biacore. Affinity determinations of the scFv antibody for zilpaterol were carried out using 'realtime' biomolecular interaction analysis. A recombinant form of PSA was also produced and characterised. Commercial anti-PSA antibodies were used to generate a competitive ELISA