The range of banned performance enhancing drugs in sports is rapidly expanding and development of detection methods does not keep up the pace with their demand. One of the drawbacks of the existing antidoping methods is the high specificity, which enables detection of common agents but not their analogs or mimetics. Consequently, the development of analytical methods is time consuming and their daily use in doping detections is expensive. The diversity of abused drugs requires the use of advanced bioanalytical procedures from the fields of biotechnology and molecular medicine. The aim of this master thesis is to design and construct probes for detection of erythropoietin receptor agonists in biological samples. For this purpose, short peptide ligands of erythropoietin receptor have been displayed on phage. The gene encoding peptide erythropoietin mimetic EMP1 was subcloned into M13KBE phage display vector. Foreign peptide was fused to the minor capsid protein p3 and displayed on all five copies on the tip of the phage. Similarly, we displayed three erythropoietin mimetic peptides (EMP1, EMP5 and AF11154) on phage in a monovalent seting using the pIT2 phagemid vector. Using enzyme-linked immunosorbent assay (ELISA) we confirmed phage binding to recombinant erythropoietin receptor ectodomain (EpoR), where, as expected, AF11154 ligand showed the lowest affinity. Recombinant phages were assessed as detections probes by concurrent incubation with a synthetic agonist or recombinant erythropoietin (rhEPO) and monitoring competive binding to immobilized EpoR. The rhEPO and synthetic agonist most effectively inhibited the binding of phages with displayed AF11154 to the receptor. By using competitive ELISA assay, we estimated the range of proportional reduction in binding of bacteriophages as a function of rhEPO concentration in the sample. This range showed to be higher than physiological concentrations of rhEPO.
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