Aptamers: a new strategy for the detection of peptide and protein doping agents in the racehorse

Improvements in biotechnology have broadened the range of potential doping agents in horse racing to include peptide hormones and proteins. Doping control laboratories require rapid, reliable and cost-effective detection methods to unambiguously identify an occurrence of protein-based drug doping. Current methods rely on high cost immunoaffinity purification with mass spectrometry detection. Aptamers are small synthetic oligonucleotides offering distinct advantages compared to antibodies in terms of thermal stability, chemical synthesis and the ability to regenerate their 3-D structure. The aims of this thesis were to examine different enrichment techniques and assess the viability of aptamers as an alternative enrichment strategy to antibodies for the detection of selected peptides and proteins. The research focused on gonadotropin releasing hormone (GnRH), insulin-like growth factor-1 (IGF-1) and analogues, recombinant human erythropoietin (rhEPO) and darbepoietin. The study of GnRH allowed for a direct comparison between a well-characterised GnRH aptamer and an established antibody-based enrichment protocol. Reliable enrichment and quantification in equine urine was achieved at 1 pg/mL. A 1 h detection window was determined from analysis of equine urine following GnRH administration. The identification of an equine catabolite, GnRH(5-10), extended this window to 4 h. Application of the aptamer enrichment technique to IGF-1 and three analogues in equine plasma facilitated the assessment of aptamer enrichment on larger, more complex proteins. A dual digestion strategy using trypsin and endoproteinase Asp-N allowed differentiation of the four proteins. The method used a characterised aptamer and reliable detection of all proteins was achieved at 25-50 ng/mL. Extension of the aptamer-based enrichment to the glycosylated protein, rhEPO, in equine plasma using an uncharacterised aptamer resulted in poor recoveries. Surface plasmon resonance analysis of the biotin-tagged EPO aptamer indicated that the tagging procedure altered the aptamer’s binding affinity. Careful characterisation of the aptamer is necessary prior to implementation as an enrichment reagent. Collectively, the findings presented in this thesis explore various enrichment strategies including SPE, immunoaffinity and aptamer-based protocols targeted at selected doping agents. This work has established a foundation for the future investigation of aptamers as a new analytical approach for the identification of peptide or protein doping in horse racing