Evaluation of methods for purifying erythropoietin from urine using immunoaffinity applications

Abstract

Erythropoietin (EPO), a hormone that regulates the synthesis of red blood cells, is frequently abused by athletes. Sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE) is an essential analytical technique in all anti-doping laboratories in order to detect the abuse of EPO. An immunoaffinity purification step is now considered essential for the pre-treatment of urine samples to isolate EPO prior to gel electrophoresis. In this study, we have compared the performance of two immunoaffinity purification techniques in EPO anti-doping analysis, i.e., the anti-EPO micro well plate and anti-EPO monolith column. The anti-EPO monolith column is efficient in removing undesirable proteins except for the Bovine Serum Albumin (BSA) as seen on SDS-PAGE. The BSA was eventually removed from the protocol and the undesirable protein band was eliminated without affecting the performance of the method. Throughout the study, the anti- EPO monolith column emerged as a better option, as it provided a higher sensitivity and higher throughput analysis when compared to the anti-EPO micro well plate. The anti-EPO monolith column has shown consistent results with the EPO recovery rate of 72%, while the limit of detection is as low as 0.5 mIU mL -1.