Abstract des Autors

Erythropoiesis Stimulating agents (ESAs), including darbepoietin (dEPO, NESP), recombinant erythropoietin(rEPO and biosimilars), EPO-Fc and methoxypolyethylene glycol-epoetin beta (CERA), are listed in the classS2 on the World Anti-Doping Agency (WADA) prohibited list of substances. ESAs, engineered for the treatmentof anaemia, are used mainly in endurance sports to increase athletic performance. The detection of their abuseby anti-doping control laboratories is performed using traditional Western blotting following either size-basedseparation (SDS/SAR-PAGE) or isoelectric focusing (IEF). Upon its introduction in the anti-doping control fightaround 2001, the EPO detection test has been continually improving. Many of the changes were realized onthe sample preparation steps, but only few modifications were made in the electrophoresis and Westernblotting procedures. The ESA testing technique is still hampered by the high cost and labor-intensive workassociated to electrophoresis. Many different studies published by academic or industrial groups have shownthat a capillary electrophoresis western system from the company ProteinSimple could produce results withhigh specificity, sensitivity and precision. Simple Western is fully automated and easily standardized, makingthe results more reproducible. The assay takes place in an ultrathin nano-capillary (5 cm x 100 μm, 400 nL)where the proteins are separated by electrophoresis, then immobilized to the capillary wall via photo-chemistryand finally probed with specific primary and secondary HRP-linked antibodies. Target detection is performedby chemiluminescence and the signal is directly detected and quantified. The system is gel-free, blot-free andall steps following the initial sample preparation do not require manual operations which increasereproducibility. During the development of the method, more than 75 runs were performed with the system, 17primary antibodies and 6 secondary antibodies were screened, 3 different loading buffers were evaluated anddifferent blocking agents were also used. More than 100 individual urine samples from athletes wereevaluated. The major breakthrough was the discovery of a biotinylated polyclonal goat anti-EPO antibody(BAF959, RnD Systems), that used in combination with a proprietary streptavidin-HRP conjugate, allowed thistechnique to be sensitive enough to believe that it could be used for initial testing procedure. The resultsobtained so far with one of the platform (WES system) demonstrated the ability of the system to correctlydiscriminate recombinant erythropoietins (EPO) from the endogenous form but more work needs to be done tofind the best analytical conditions.