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.

中文翻译：

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兴奋剂分析中促红细胞生成素的SAR-PAGE测试的比较和优化。

促红细胞生成素（EPO）是世界反兴奋剂机构（WADA）禁用清单S2中列出的物质，运动员通常使用它们来提高耐力表现。根据当前的WADA技术文件，肌氨酰聚丙烯酰胺凝胶电泳（SAR-PAGE）然后进行蛋白质印迹以根据其分子量区分促红细胞生成素是唯一可用于筛选和确认所有类型促红细胞生成素的方法。免疫纯化和蛋白质转移的效率对于确保促红细胞生成素检测的选择性和灵敏度至关重要。这项研究对SAR-PAGE测试进行了一些比较和优化。我们优化了第一个印迹条件，然后根据其选择性，可重复性，以及尿液和血液分析的灵敏度。此外，建立并比较了尿液和血液分析的快速程序。1.0 mA / cm下的两步操作与通常使用的恒定电流方法相比，使用²持续60分钟，然后1.56 mA / cm ²持续20分钟，可以提高吸印效率。免疫纯化的比较表明，不同方法之间的选择性和敏感性没有显着差异。对于其他因素，例如操作复杂性，时间和成本，建议分别使用StemCell®纯化试剂盒，单印迹和磁珠再进行双印迹进行尿液筛查和确认。虽然建议分别使用磁珠和MAIIA®试剂盒，然后再进行两次印迹法同时进行血样的筛查和确认。为了确保高灵敏度和选择性，建议使用双印迹法对尿液和血液进行快速分析。