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

Abstract

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.