Cost Minimized Immunoaffinity Purification of EPO and Its Analogs in Doping Control-A Step-by-Step Protocol for Human Urine and Blood.

IF 2.6 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Drug Testing and Analysis Pub Date : 2025-01-27 DOI:10.1002/dta.3848

Christian Reichel, Günter Gmeiner, Mario Thevis

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引用次数: 0

Abstract

A cost minimized immunoaffinity protocol was developed, which allows the direct purification of ERAs (urinary and recombinant human EPO, Darbepoetin, EPO-Fc, CERA) from human urine. The method applies magnetic beads and needs no covalent immobilization of the capture antibody. It requires only 10 mL of urine, 1 μg of anti-EPO antibody, and 25 μL of bead slurry. The beads are coated with the capture antibody in advance and can be stored in the refrigerator for months without any loss of functionality. The protocol was fully validated in combination with SAR-PAGE and Western blotting using the biotinylated clone AE7A5 anti-EPO antibody. It is compliant with the criteria of TD2024EPO of the World Anti-Doping Agency (WADA) for the gel electrophoretic detection of ERAs. For each ERA, the achieved limit of detection (LOD) is at least one tenth of the minimum required performance limit (MRPL) of WADA, that is, 0.1 IU/L, 0.1 pg/mL, 0.5 pg/mL, and 0.5 pg/mL for rEPO, Darbepoetin, EPO-Fc, and CERA, respectively. After slight modification, the protocol is also applicable to serum and plasma and also fulfils the corresponding MRPLs of WADA for these matrices. Compared to commercial immunoaffinity purification kits for EPO, the material costs are significantly lower but with identical results.

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我们开发出了一种成本最低的免疫亲和方案，可直接从人体尿液中纯化ERAs（尿液和重组人EPO、Darbepoetin、EPO-Fc、CERA）。该方法采用磁珠，无需共价固定捕获抗体。只需 10 mL 尿液、1 μg 抗 EPO 抗体和 25 μL 磁珠浆液。微珠事先涂有捕获抗体，可在冰箱中保存数月而不会失去功能。该方案结合使用生物素化克隆 AE7A5 抗EPO 抗体的 SAR-PAGE 和 Western 印迹法进行了充分验证。该方案符合世界反兴奋剂机构（WADA）TD2024EPO 关于ERA凝胶电泳检测的标准。对于每种ERA，达到的检测限（LOD）至少是世界反兴奋剂机构最低要求性能限（MRPL）的十分之一，即rEPO、Darbepoetin、EPO-Fc和CERA分别为0.1 IU/L、0.1 pg/mL、0.5 pg/mL和0.5 pg/mL。稍作修改后，该方案也适用于血清和血浆，并符合世界反兴奋剂机构对这些基质的相应 MRPLs 要求。与 EPO 的商业免疫亲和纯化试剂盒相比，材料成本大大降低，但结果完全相同。

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来源期刊

Drug Testing and Analysis BIOCHEMICAL RESEARCH METHODS-CHEMISTRY, ANALYTICAL

CiteScore

5.90

自引率

24.10%

发文量

191

审稿时长

2.3 months

期刊介绍： As the incidence of drugs escalates in 21st century living, their detection and analysis have become increasingly important. Sport, the workplace, crime investigation, homeland security, the pharmaceutical industry and the environment are just some of the high profile arenas in which analytical testing has provided an important investigative tool for uncovering the presence of extraneous substances. In addition to the usual publishing fare of primary research articles, case reports and letters, Drug Testing and Analysis offers a unique combination of; ‘How to’ material such as ‘Tutorials’ and ‘Reviews’, Speculative pieces (‘Commentaries’ and ‘Perspectives'', providing a broader scientific and social context to the aspects of analytical testing), ‘Annual banned substance reviews’ (delivering a critical evaluation of the methods used in the characterization of established and newly outlawed compounds). Rather than focus on the application of a single technique, Drug Testing and Analysis employs a unique multidisciplinary approach to the field of controversial compound determination. Papers discussing chromatography, mass spectrometry, immunological approaches, 1D/2D gel electrophoresis, to name just a few select methods, are welcomed where their application is related to any of the six key topics listed below.