Multiplexed detection of agents affecting erythropoiesis (AAEs) and overall strategy for optimizing initial testing...

Area of Science:

• Sports Science
• Biochemistry
• Analytical Chemistry

Background:

• Erythropoietin receptor agonists (ERAs) are prohibited doping agents targeting early erythropoiesis.
• Luspatercept, a novel transforming growth factor-b inhibitor, promotes later-stage erythropoiesis and poses a potential doping risk.
• Synergistic effects between Luspatercept and recombinant erythropoietin (rEPO) may enhance red blood cell production.

Purpose of the Study:

• To develop a combined detection method for ERAs and Luspatercept.
• To maintain or improve the sensitivity and specificity of existing ERA detection methods.
• To reduce analysis time and sample volume in anti-doping laboratories.

Main Methods:

• Utilized magnetic beads coated with antibodies for sample immunoprecipitation, enabling multiplexing.
• Employed Surface Assay/SDS-PAGE (SAR/SDS-PAGE) as authorized by the World Anti-Doping Agency (WADA).
• Incorporated biotinylated primary antibodies for sensitive and specific immunodetection.

Main Results:

• Developed a robust method for the simultaneous detection of agents affecting erythropoiesis (AAEs).
• The method demonstrated high specificity and sensitivity.
• The assay is easily and quickly implementable in anti-doping laboratories.

Conclusions:

• The developed method effectively detects both ERAs and Luspatercept.
• This technique enhances anti-doping strategies against novel performance-enhancing substances.
• The method is suitable for widespread adoption by anti-doping laboratories worldwide.