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.

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.