Erythropoietin is a protein whose main function is stimulation of erythropoiesis, which is why recombinant human erythropoietins are commonly misused as performance enhancing drugs by some athletes. It is suspected that numerous novel erythropoietin mimetics structurally unrelated to endogenous erythropoietin are also being used. Most of the modern anti-doping methods fail to detect such substances, as they mostly rely on detection of structurally well known substances or, more inconveniently, on the prolonged control of certain parameters in athlete biological specimens. Because of this it is the aim of this master’s thesis to establish and evaluate a novel universal assay platform which would allow the detection of all erythropoietin mimetics in biological specimens, regardless of their structure. The assay platform is based on recombinant filamentous phage particles displaying the erythropoietin receptor ectodomain (EpoR), which are used as detection probes instead of primary antibodies in two distinct immunoassays (ELISAs). ELISA1, with immobilized EpoR ligands detects phages with unoccupied EpoR whereas ELISA2, with immobilized anti-c-myc antibodies, reflects the proportion of phages with occupied EpoR. The ELISA1/ELISA2 signal ratio suggests the presence (ratio greater than 1) or absence (ratio less than 1) of erythropoietin mimetics in the specimen. Following the preparation of recombinant phage particles and the evaluation of the functionality of the displayed EpoR, we determined the optimal concentrations of phages, antibodies and EpoR ligands for further use in the assay platform. The preliminary results of the assay platform using artificial matrices either spiked with erythropoietin mimetic (“suspicious”) or intact (“clean”), showed that while ELISA1 was sensitive enough to discriminate between clean and suspicious samples, the signals in ELISA2 were unexpectedly high regardless of the analyzed sample. After testing the efficacy of phage depletion using paramagnetic beads we came to the conclusion that the recombinant phages were displaying myc-tag without a functional EpoR. We hypothesized, and later confirmed by western blot, that the reason for this anomaly is the proteolytic cleavage of the displayed fusion protein, which leaves myc-tag intact and removes the functional EpoR. Consequentially, a significant number of phages bind to the immobilized anti-c-myc antibodies in ELISA2 even without displaying EpoR. For further optimization of the platform we recommend relocating the myc-tag to the N-terminus of the displayed EpoR.
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