Recombinant erythropoietin(Biogen (Denmark) A/S)

Optimal detection of Erythropoietin receptor agonists (ERAs) in anti-doping analyses relies heavily on efficient immunopurification (IP) strategies. While antibody-based IP is routinely applied, aptamers have emerged as promising alternatives. This proof-of-principle study investigated the applicability of an anti-EPO DNA aptamer for ERA enrichment in serum, followed by Sarkosyl-Polyacrylamide Gel Electrophoresis (SAR-PAGE) analysis. The aptamer-based method successfully captured recombinant ERA variants (rEPO, dEPO, EPO-Fc, CERA) with defined limits of detection (LOD) in serum: 10 mIU/mL for rEPO, 10 pg/mL for dEPO, and 25 pg/mL for EPO-Fc and CERA. In comparison, the antibody-based method achieved lower serum LODs of 5 mIU/mL for rEPO, 5 pg/mL for dEPO, and 12.5 pg/mL for EPO-Fc and CERA. Both methods demonstrated good selectivity, as no non-specific bands were detected in blank serum matrices. However, endogenous blood EPO (bEPO) was not enriched by the aptamer, likely reflecting glycosylation-related structural differences between endogenous and recombinant isoforms. These findings highlight that aptamers can provide highly specific recognition of recombinant ERAs without cross-reactivity to bEPO, which may simplify interpretation in doping control analyses. Nonetheless, further optimization-such as refining immobilization chemistries, incorporating differently glycosylated EPO forms during SELEX, and sequence improvements-is required to enhance capture of native isoforms. Overall, this study establishes a foundation for aptamer-based enrichment as a complementary alternative to antibody-based methods in anti-doping applications.