促红细胞生物药物，包括darbepoietin (dEPO, NESP), recombinant erythropoietin (rEPO and biosimilars), EPO-Fc 和 methoxy polyethylene glycol-epoetin beta (CERA),被严禁在体育运动赛事中使用(世界反兴奋剂代码禁止的物质清单，S2类)。
传统western blot，毛细管电泳，SDS-page电泳等技术已经在兴奋剂检测中广泛使用。但是这些方法，需过多手工操作，通量低，不能准确定量，无法实现对兴奋剂的大规模定量检测。
加拿大阿曼德-弗雷皮尔研究所(INRS-Institut Armand-Frappier)的研究人员最近基于ProteinSimple毛细管电泳Western Blot技术（Simple Western）开发超敏，快速，精准，定量，高特异性的尿液和血液兴奋剂检测方法。结果发表在最新一期，Drug Test Anal. 2018 Oct 26. doi: 10.1002/dta.2528.
原文阅读：
A new approach may be useful to this end. Protein Simple (PS) offers a system, “Capillary-Electrophoresis Western blotting (Simple Western)” that was described as showing specificity, sensitivity and precision required for testing protein targets in the low picogram levels in different fields of application 11-15. According to the Protein Simple, the assay takes place in an ultrathin nano-capillary (5 cm x 100 μm, 400 nL) where the proteins are first separated by electrophoresis, then immobilized to the capillary wall via a photo-chemical reaction and finally probed with specific primary and secondary HRP-linked antibodies. Target detection is performed by chemiluminescence and the signal is directly quantified. The system is gel-free, blot-free and all steps following the initial sample preparation do not require manual operations which increases the reproducibility of the assay 16. If proven to be sufficiently sensitive and specific, this new automated immunoassay technique could simplify the analysis of ESAs in urine and blood. Furthermore, the platform is fully automated and thus this technology could be easily implemented in anti-doping control laboratories, which would contribute to the improvement of the overall assay performance and standardization of the method. With reduced sample preparation and analysis, such an automated system could be of interest during major sports events such as the Olympic Games where a high number of samples need to be analyzed in a short period of time.
样品：
运动员尿液样品及将加入标注品的正常尿液
1）Anonymized athletes’ urine samples received for testing (from athletes having signed consent for having their samples used for research purposes) that were reported previously as negative or positive, were reanalysed in this study
2）Once the conditions were established to give reproducible results with pure reference standards solutions, negative (blank) urine samples were spiked with the standards to determine if it was possible to discriminate the ESAs from huEPO
抗体选择
3. 结果
多种兴奋剂检测
PNGase去除N-glycan对分子量的影响
不同抗体稀释度对检出结果的影响
确定NESP和EPO LOD
通过Spike标准品样品确认信号特异性
运动员尿液样本检测
Samples at position 3, 5, 6, 7, 9 and 10 showed a mixed EPO profile while samples at position 1, 2, 4, and 8 had profiles closer to the reference standard with MW range of 60-62 kDa, indicating that they contain mainly rhEPO.
更多Simple Western技术，请联系ProteinSimple中国
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