Cost Minimized Immunoaffinity Purification of EPO and Its Analogs in Doping Control-A Step-by-Step Protocol for Human Urine and Blood.

Abstract

A cost minimized immunoaffinity protocol was developed, which allows the direct purification of ERAs (urinary and recombinant human EPO, Darbepoetin, EPO-Fc, CERA) from human urine. The method applies magnetic beads and needs no covalent immobilization of the capture antibody. It requires only 10 mL of urine, 1 μg of anti-EPO antibody, and 25 μL of bead slurry. The beads are coated with the capture antibody in advance and can be stored in the refrigerator for months without any loss of functionality. The protocol was fully validated in combination with SAR-PAGE and Western blotting using the biotinylated clone AE7A5 anti-EPO antibody. It is compliant with the criteria of TD2024EPO of the World Anti-Doping Agency (WADA) for the gel electrophoretic detection of ERAs. For each ERA, the achieved limit of detection (LOD) is at least one tenth of the minimum required performance limit (MRPL) of WADA, that is, 0.1 IU/L, 0.1 pg/mL, 0.5 pg/mL, and 0.5 pg/mL for rEPO, Darbepoetin, EPO-Fc, and CERA, respectively. After slight modification, the protocol is also applicable to serum and plasma and also fulfils the corresponding MRPLs of WADA for these matrices. Compared to commercial immunoaffinity purification kits for EPO, the material costs are significantly lower but with identical results.