Easy-to-use IEF compatible immunoaffinity purification of Erythropoietin from urine retentates.

Erythropoietin (EPO) is a peptide hormone responsible for hypoxia-induced promotion of erythrocyte production. The possibility of enhancing oxygen transport through an increase of erythrocytes has led to EPO abuse in sports. Detection of exogenous EPO is most commonly done via isoelectric focusing (IEF) which is a method provided by the Technical Document TD2009EPO of the World Anti-Doping Agency (WADA). Before analysis, collected urine samples need to be concentrated 500- to 1000-fold, leading to high protein abundance in the retentates. Reduction of protein concentration through an immunoaffinity purification using ELISA wells has been successfully used prior to SDS-PAGE. This ELISA kit was used to purify samples using an IEF-compatible elution. The purification showed recovery ratios between 50 and 90% depending on substance and application volume. Application of immunopurified samples to IEF was shown to improve the quality of the gels by reducing streaks and curvatures within the lanes and bands of the gel. The result was an increase of quality for IEF gels.

Effects of high intensity exercise on isoelectric profiles and SDS-PAGE mobility of erythropoietin.

Exercise induced proteinuria is a common phenomenon in high performance sports. Based on the appearance of so called "effort urines" in routine doping analysis the purpose of this study was to investigate the influence of exercise induced proteinuria on IEF profiles and SDS-PAGE relative mobility values (rMVs) of endogenous human erythropoietin (EPO). Twenty healthy subjects performed cycle-ergometer exercise until exhaustion. VO (2)max, blood lactate, urinary proteins and urinary creatinine were analysed to evaluate the exercise performance and proteinuria. IEF and SDS-PAGE analyses were performed to test for differences in electrophoretic behaviour of the endogenous EPO before and after exercise. All subjects showed increased levels of protein/creatinine ratio after performance (8.8+/-5.2-26.1+/-14.4). IEF analysis demonstrated an elevation of the relative amount of basic band areas (13.9+/-11.3-36.4+/-12.6). Using SDS-PAGE analysis we observed a decrease in rMVs after exercise and no shift in direction of the recombinant human EPO (rhEPO) region (0.543+/-0.013-0.535+/-0.012). Following identification criteria of the World Anti Doping Agency (WADA) all samples were negative. The implementation of the SDS-PAGE method represents a good solution to distinguish between results influenced by so called effort urines and results of rhEPO abuse. Thus this method can be used to confirm adverse analytical findings.

Discrimination of recombinant and endogenous urinary erythropoietin by calculating relative mobility values from SDS gels.

Erythropoietin (EPO) promotes the production of red blood cells, the key factor in the regulation of the oxygen transport, and has been abused by athletes for performance enhancement in endurance sports. Current methods to detect EPO misuse are based on isoelectric focussing (IEF), double blotting, and chemiluminescence detection. A new approach utilizing SDS-PAGE mobilities of target analytes is presented. Employing two internal standards (novel erythropoiesis stimulating protein and recombinant rat EPO), the assay provides a tool which allows the calculation of relative mobility values for endogenous urinary EPO and recombinant epoetins (e.g., Dynepo) and, thus, the distinction of these analytes in doping control samples. A reference group of 53 healthy volunteers and samples originating from a Dynepo (epoetin delta) excretion study conducted with a single person were analyzed and led to a significant discrimination of endogenous urinary and recombinant EPO. A clear differentiation was accomplished over a period of four days post-administration of a single injection of 50 IU/kg body weight. Hence, the method may be useful as a screening procedure in doping control or as complementary confirmation tool to the established IEF assay.