Comparison and optimization of SAR‐PAGE tests for erythropoietins in doping analysis

Abstract

Erythropoietins (EPOs) are substances listed in S2 of the World Anti‐Doping Agency (WADA) Prohibited List and are used commonly by athletes to increase endurance performance. According to the current WADA Technical Documents, sarcosyl‐polyacrylamide gel electrophoresis (SAR‐PAGE) followed by western blotting to differentiate erythropoietins based on their molecular weights is the only method that can be used for both screening and confirmation of all types of erythropoietins. The efficiency of immunopurification and protein transfer is crucial for ensuring the selectivity and sensitivity of erythropoietin detection. Several comparisons and optimization of the SAR‐PAGE tests were conducted in this study. We optimized the first blotting conditions and then compared different immunopurification methods based on their selectivity, repeatability, and sensitivity for both urine and blood analysis. Additionally, rapid procedures for both urine and blood analysis were established and compared. The two‐step procedure at 1.0 mA/cm2 for 60 min followed by 1.56 mA/cm2 for 20 min increased the blotting efficiency compared with the commonly used constant current approach. Comparison of immunopurification revealed no significant difference in selectivity and sensitivity between the different methods. For other factors, such as operation complexity, time and cost, a StemCell® purification kit followed by single blotting and magnetic beads followed by double blotting are recommended for urine screening and confirmation, respectively. While magnetic beads and a MAIIA® kit followed by double blotting are recommended for both screening and confirmation of blood samples, respectively. To ensure high sensitivity and selectivity, double blotting is recommended for a rapid procedure for both urine and blood analysis.

Research on spiking rat EPO as internal standard in doping control samples for detection of EPO using SAR‐PAGE analysis with biotinylated primary antibody

“…However, although CERA was detected in all 18 urine samples, the signal intensity of the band in different urine samples was observed to be erratic (Figure 4A). This phenomenon also occurred in our previous study when the StemCell® ELISA plate or the magnetic beads method were used for immunopurification of urine samples, and we assumed that the reason behind this might be the presence of proteins in urine samples, which might prevent the interaction between the antibody and CERA 11 …”

“…For the evaluation of the repeatability, all EPO and its analogs were spiked into blank urine or serum samples at amounts around their estimated LOD, except for CERA for urine samples. Based on previous research, 11 as the recovery of CERA in different urine samples was shown to be erratic, the amounts of spiked CERA for urine samples were much higher than its estimated LOD. Each blank urine sample was spiked with BRP (2.5 mIU), NESP (25 pg), EPO‐Fc (25 pg), and CERA (500 pg).…”

“…Validation of the methods incorporated aspects of selectivity, repeatability, and sensitivity. The experimental procedure was conducted as mentioned previously, 11 with minor modifications.…”

“…SAR‐PAGE was conducted at a constant voltage of 200 V for 75 min. A two‐step blotting procedure was applied after SAR‐PAGE separation 11 . The first step applied an electric current of 1.0 mA/cm 2 for 60 min, followed by 1.56 mA/cm 2 for 20 min.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…Obtained analytical results were similar compared with other IA‐based sample preparation options, and the presented approach offered a rapid and simple alternative to other strategies based on, for example, magnetic nanoparticles or ELISA microplates. Zhou et al also elaborated on the critical role of the first IA‐based sample preparation step as well as the efficiency of the first blot for optimal analytical test results and recommended the use of ELISA microplates combined with a two‐step blotting protocol (1 hr at 1.0 mA/cm 2 plus 20 min at 1.56 mA/cm 2 ) instead of a commonly used single constant current approach for initial testing purposes, allowing for substantially reduced turnaround times for analytical test results 104 . For confirmatory analyses, IA‐assisted analyte extraction with antibody‐coated magnetic nanoparticles prior to gel electrophoresis and double blotting was advised.…”