A new principle suggested for detection of darbepoetin-alpha (NESP) doping

Doping with darbepoetin-alpha, also termed novel erythropoiesis stimulating protein (NESP), a hypersialylated, very effective analogue of erythropoietin, is a serious threat in sport. We report here on a new principle for the detection of darbepoetin-alpha in serum based upon increase in immunoactivity after desialylation with neuraminidase. The method is evaluated on sera from patients taken 2-14 days after last injection of darbepoetin-alpha. Thirty-two venous blood samples and 3 capillary samples taken from finger tips were obtained from 13 patients with end stage renal disease treated with intravenous or subcutaneous injections of Aranesp, 0.45 to 2.60 microg*kg(-1). Blood samples from 37 individuals with endogenous erythropoietin were used as controls. The sera were diluted 1:2 with acetate buffer pH 5.6 with or without neuraminidase and incubated at 37 degrees C for 1 or 24 h before immunoassay. The erythropoietin immunoactivity in serum volumes of 12.5-50 microL was measured with ELISA-kits from R&D Systems Inc and medac GmbH. The relative increase in immunoactivity after desialylation was in all cases higher for the darbepoetin-alpha samples than for any of the control samples assayed in parallel, varying incubation time with the enzyme, serum volumes and batches of both ELISA-kits. The mean relative increase in immunoactivity of endogenous erythropoietin after neuraminidase was 42% with the medac-kit and 117% with R&D-kit while the corresponding figures for darbepoetin-alpha were 282% and 231% with 1 h and 299% and 256% with 24 h enzyme incubation, respectively. Endogenous and recombinant human erythropoietin showed similar relative increase after desialylation. The method to detect darbepoetin-alpha in serum is simple to perform, robust, sensitive and requires a small amount of blood. The drug was detected in all patient sera taken 2-14 days after last injection. We suggest that the method should be evaluated for the detection of darbepoetin-alpha doping in sport.