Development of an Enzyme‐Linked Immunosorbent Assay for Pegmolesatide in Doping Analysis: An Initial Testing Method

ABSTRACT

Pegmolesatide is a novel synthetic erythropoietin‐mimetic agent that binds to and activates the erythropoietin receptor (EPOR), thereby stimulating erythropoiesis. Owing to its erythropoiesis‐stimulating activity, it has been included in the latest World Anti‐Doping Agency (WADA) Prohibited List as an erythropoietin receptor agonist (S2.1.1). The current analytical approach for pegmolesatide in doping analysis mainly relies on bottom‐up nano‐liquid chromatography–high‐resolution tandem mass spectrometry (LC‐HRMS) in serum. Although accurate and sensitive, this approach involves extensive sample preparation and time‐consuming LC analysis, which limits its suitability for high‐throughput initial testing procedure (ITP). In this study, an enzyme‐linked immunosorbent assay (ELISA), termed the EPOR–anti‐PEG ELISA, was developed as a screening method for pegmolesatide in urine and serum. The assay employs EPOR as the capture reagent to enrich pegmolesatide, followed by detection of its polyethylene glycol (PEG) moiety using the anti‐PEG monoclonal antibody RM105. Method validation included assessment of selectivity, limit of detection (LOD), dynamic range, reliability, and carryover. Under optimized conditions, the assay showed an LOD of 1 ng/mL in urine, with an adjusted OD ₄₅₀ screening cutoff of 0.029. In serum, with an adjusted OD ₄₅₀ screening cutoff of 0.243, the LOD was 2 ng/mL, which was lower than the limit of identification (LOI) of 7 ng/mL determined by LC‐HRMS. Overall, this ELISA provides a streamlined, cost‐effective, and high‐throughput screening approach for pegmolesatide and may serve as a practical complement to the LC‐HRMS method in doping analysis.