Abstract

This research describes the development of immunoassays for the detection of residues of three anti-protozoan drugs (coccidiostats), namely, halofuginone,toltrazuril and diclazuril, used in the treatment of Eimeria spp.infections in cattle,pigs, chickens and turkeys. An avian-derived halofuginone-specific scFv library was constructed, from which the best scFv isolated did not possess the required analytical sensitivity. To circumvent this, genetic engineering via light-chain shuffling of the optimal light:heavy chain pairings of the scFv library were investigated. Use of this light-chain shuffled library and rigorous screening, led to the isolation of a scFv with a 185-fold greater sensitivity than the original. This scFv was incorporated into both enzyme-linked immunosorbent assay (ELISA) and Biacore assay formats that were applied successfully for the detection of halofuginone in ‘spiked’ egg samples. Immune phage display libraries were constructed and screened for diclazuril using a number of diclazuril-conjugates. A functional diclazuril-specific scFv could not be isolated, due to complications which arose during library amplification.Subsequently, an anti-diclazuril polyclonal antibody was sourced externally, characterised and used in the development of a high sensitivity lateral-flow immunoassay for diclazuril detection in eggs. Rabbit polyclonal antibodies to the primary metabolite of toltrazuril, ‘ponazuril’,were produced, characterised and were subsequently used to generate ELISA,lateral-flow and Biacore-based assays. The performance of each of these assay formats for the detection of toltrazuril in ‘spiked’ egg samples was directlycompared. Finally, multi-coccidiostat detection in a lateral-flow dipstick format, for the simultaneous determination of halofuginone, diclazuril and toltrazuril in eggs, using antibody-coated coloured latex particles as the detection element, was successfully optimised.