The research presented in this thesis describes the production and application of antibodies against morphine-3-glucuromde and parathion, an organophosphate pesticide.
A variety of morphine-3-glucuronide-protein conjugates were produced and used to generate and characterise polyclonal antibodies. These 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’).
Combinatorial single chain Fv (scFv) antibody phage display libraries were also generated to morphine-3-glucuromde Splenomic mRNA from mice pre-immunised with a morphine-3-glucuronide-BSA conjugate was used in the amplification of antibody genes followed by cloning into vectors from a well-established phage display system. Two positive clones were isolated in the first two rounds of panning. One clone (E3) was selected and re-cloned into a plasmid for 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 E3 clone was carried out in the development of an inhibition BIAcore assay for morphine-3-glucuromde in biological matrices Affinity determinations of the scFv antibody to morphine-3- glucuromde were carried out using ‘real-time’ biomolecular interaction analysis.
Polyclonal antibodies to the organophosphate pesticide, parathion, were also produced and characterised. They were used to generate competitive ELISA and BIAcore-based inhibition immunoassays However, when analysed on the BIAcore, the parathion polyclonal antibody displayed no inhibition.