Research on Spiking Rat EPO as Internal Standard in Doping Control Samples for Detection of EPO using SAR‐PAGE Analysis with Biotinylated Primary Antibody

: According to the current Technical Document (TD) for erythropoietin (EPO), SAR-PAGE is the most commonly applied method for both screening and confirmation procedures. Although this method is effective and robust, it lacks an internal standard (IS) to monitor the efficiency of analysis for each sample covering every step of the whole procedure, including preparation, immunopurification, and western blotting. This IS needs to be recognized by both anti-EPO antibodies used for immunopurification and western blotting, respectively. Besides that, the band of IS could not be allowed to interfere with the recognition of all types of targeted EPO and analogs. To meet these two principles, rat EPO was selected. In this study, rat EPO was used to spike both urine and blood samples at the beginning of analysis. After preparation and immunopurification, we performed single blotting with biotinylated AE7A5 as the primary antibody, followed by incubation with streptavidin-coupled HRP. Based on the comparison of different immunopurification methods, the AB-286-NA antibody coupled to M-280 magnetic beads was the better choice for urine samples, whereas the MAIIA column was suitable for blood samples. All these methods were validated for selectivity, repeatability, and sensitivity. The modified method in this study could not only eliminate the cross-reactivity between antibodies but also monitor the whole procedure of the analysis of EPO with spiked rat EPO. Besides that, rat EPO could also be used as an indicator for monitoring the presence of protease(s) in urine samples.