Horseradish‐peroxidase‐conjugated anti‐erythropoietin antibodies for direct recombinant human erythropoietin detection: Proof of concept,Drug Testing and Analysis

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Horseradish‐peroxidase‐conjugated anti‐erythropoietin antibodies for direct recombinant human erythropoietin detection: Proof of concept
Drug Testing and Analysis ( IF 2.6 ) Pub Date : 2020-10-29 , DOI: 10.1002/dta.2957
Sven C Voss ₁ , Nelson N Orie _{1,

2} , Wesal El-Saftawy ₁ , Stephanie Saghbazarian ₁ , Alanoud Al-Kaabi ₁ , Costas Georgakopoulos ₁ , Ioanna Athanasiadou ₁ , Vidya Mohamed-Ali _{1,

2} , Mohammed Al-Maadheed _{1,

2}

Affiliation

Antidoping testing for recombinant human erythropoietin (EPO) is routinely performed by gel electrophoresis followed by western blot analysis with primary and secondary antibodies. The two antibody steps add more than 24 h to the testing time of a purified sample. The aim of this study was to test the concept of using directly horseradish‐peroxidase (HRP)‐conjugated anti‐EPO primary antibody, without the need for a secondary antibody, to reduce the analysis time and eliminate non‐specific cross‐reactivity with secondary antibodies. An in‐house, periodate coupling (R&D systems, clone AE7A5) and three commercially available anti‐human EPO‐HRP conjugates from Genetex, Novus Biologicals and Santa Cruz were evaluated for specificity and sensitivity, using recombinant human EPO standards, negative human urine samples and urine samples from an EPO excretion study. The in‐house anti‐EPO‐HRP conjugate was performed as well as the current two‐step application of unconjugated primary and secondary antibodies used in routine analysis, with comparable specificity and sensitivity. The analysis time was markedly reduced for purified samples from 25 h with the routine method down to 7 h with the in‐house HRP conjugate. Of the three commercially available conjugates tested, only the Santa Cruz anti‐EPO‐HRP conjugate showed comparable specificity but had lower sensitivity to both the in‐house and the antibody combination currently applied routinely. The other two commercially available conjugates (Genetex and Novus Biologicals) did not show any visible bands with the EPO standards. The results clearly demonstrate the potential utility of a directly HRP‐conjugated anti‐EPO antibody to reduce analysis time for EPO in doping control.

中文翻译：

用于直接检测重组人促红细胞生成素的辣根过氧化物酶偶联抗促红细胞生成素抗体：概念验证

重组人促红细胞生成素 (EPO) 的抗兴奋剂检测通常通过凝胶电泳进行，然后用一抗和二抗进行蛋白质印迹分析。这两个抗体步骤使纯化样品的测试时间增加了 24 小时以上。本研究的目的是测试直接使用辣根过氧化物酶 (HRP) 偶联的抗 EPO 一抗的概念，无需二抗，以减少分析时间并消除与二抗的非特异性交叉反应。抗体。使用重组人 EPO 标准品评估了一种内部高碘酸盐偶联物（研发系统，克隆 AE7A5）和来自 Genetex、Novus Biologicals 和 Santa Cruz 的三种市售抗人 EPO-HRP 偶联物的特异性和敏感性，来自 EPO 排泄研究的阴性人类尿液样本和尿液样本。进行了内部抗 EPO-HRP 偶联物以及当前用于常规分析的未偶联一抗和二抗的两步应用，具有相当的特异性和灵敏度。纯化样品的分析时间从使用常规方法的 25 小时显着减少到使用内部 HRP 偶联物的 7 小时。在测试的三种市售偶联物中，只有 Santa Cruz 抗 EPO-HRP 偶联物表现出相当的特异性，但对内部和目前常规应用的抗体组合的敏感性较低。其他两种市售偶联物（Genetex 和 Novus Biologicals）在 EPO 标准品中没有显示任何可见条带。

更新日期：2020-10-29

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