SARCOSYL-PAGE: a new method for the detection of MIRCERA- and EPO-doping in blood

Abstract

The detection of doping with MIRCERA (the brand name for Continuous Erythropoietin Receptor Activator, or CERA) is hampered by the limited excretion of the rather large molecule (approximately 60 kDa) in urine. Blood (serum, plasma) in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) appears to be the ideal matrix for detecting all forms of doping with erythropoiesis-stimulating agents (ESAs) because the apparent molecular masses of ESAs are different from the mass of human serum erythropoietin (shEPO). While SDS-PAGE has proven the most sensitive method for the detection of doping with Dynepo, the sensitivity of SDS-PAGE for MIRCERA is drastically decreased. By exchanging the SDS for SARCOSYL (SAR) in the sample and running buffers the sensitivity problem was solved. SARCOSYL, a methyl glycine-based anionic surfactant, is only binding to the protein-part of MIRCERA but not to its polyethylene glycol (PEG)-chain, while SDS binds to both parts. In consequence, the monoclonal anti-EPO antibody (clone AE7A5) no longer interacts with the fully SDS-solubilized MIRCERA molecules. Only those molecules that contain SDS bound to the protein-chain are detected. Due to the inability of SARCOSYL to solubilize PEG-molecules, MIRCERA can be detected on SARCOSYL-PAGE with the same sensitivity as non-PEGylated epoetins. In a typical SAR-PAGE experiment, 200 microL of serum are used, which allows the direct detection of MIRCERA, recombinant epoetins (such as NeoRecormon, Dynepo, NESP), and shEPO in a single experiment and with high (i.e. femtogram) sensitivity.