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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Sports Science

Background:

• Pegmolesatide, an erythropoietin receptor agonist, is banned in sports by the World Anti-Doping Agency (WADA).
• Current detection methods like LC-HRMS are sensitive but time-consuming and require extensive sample preparation, hindering high-throughput initial testing.

Purpose of the Study:

• To develop a rapid, cost-effective screening assay for pegmolesatide in doping analysis.
• To establish an enzyme-linked immunosorbent assay (ELISA) as a complementary method to LC-HRMS for pegmolesatide detection.

Main Methods:

• Development of an EPOR-anti-PEG ELISA using erythropoietin receptor (EPOR) as the capture reagent.
• Detection of the polyethylene glycol (PEG) moiety of pegmolesatide using the anti-PEG monoclonal antibody RM105.
• Method validation including selectivity, limit of detection (LOD), dynamic range, reliability, and carryover assessment in urine and serum.

Main Results:

• The EPOR-anti-PEG ELISA achieved an LOD of 1 ng/mL in urine and 2 ng/mL in serum.
• Screening cutoffs were established at OD450 of 0.029 for urine and 0.243 for serum.
• The developed ELISA demonstrated lower LODs than the LOI for LC-HRMS in serum, indicating enhanced sensitivity for screening.

Conclusions:

• The developed ELISA provides a streamlined, cost-effective, and high-throughput screening method for pegmolesatide.
• This ELISA serves as a practical complement to LC-HRMS, enhancing doping control capabilities.
• The assay facilitates efficient initial screening of athletes for prohibited erythropoiesis-stimulating agents.