Immunochemical analysis of illegal steroid growth promoters in cattle
Fitzpatrick, Jane (2002) Immunochemical analysis of illegal steroid growth promoters in cattle. PhD thesis, Dublin City University.
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Steroid hormones and their analogues can be used as growth promoters in cattle. The use of these substances is banned in the EU, and hence each member state must ensure that cattle are not being dosed with these drugs. This study focuses on the immunochemical detection of three steroidal growth promoters, ethynylestradiol (EE), diethylstilbestrol (DES) and trenbolone (TR).
Polyclonal antibodies were raised against EE and TR. The anti-TR antibodies were applied to two competitive ELISA formats, producing assays as sensitive as other reported systems while needing very little pre-treatment of the bile samples. The anti-EE antibodies were also applied to these ELISA systems and also used to develop a biosensor-based assay using a BIAcore 3000®. These assays allowed the detection of EE at low ng ml"1 quantities in bovine bile.
As an alternative antibody supply the production of scFv antibody fragments was investigated. Two naive phage display libraries, one murine and one human, were panned for the presence of binding fragments specific for the target steroid molecules. A number o f pre-immunised libraries were also constructed and selected against the antigens of interest. None of the approaches provided specific clones that could be used as immunoreagents in analysis.
An investigation into the reduction of the incubation time required to perform a competitive ELISA was carried out. This method was shown to be effective for some of the polyclonal antibodies tested. The successful assays were applied to a prototype rapid ELISA machine. The ability of this device to detect low quantities of steroids in both a single and mixed sample was studied. A preliminary, prototype assay for the detection of two separate steroid growth promoters in a single sample was shown.
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