Department of Physiology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara 252-0374, Japan.
Department of Nephrology, Kumamoto University Graduate School of Medical Sciences, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan.
Molecules. 2023 May 30;28(11):4446. doi: 10.3390/molecules28114446.
Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using isoelectric focusing (IEF)-PAGE to show that natural Epo and injected erythropoiesis-stimulating agents (ESAs) appear in different pH areas. Next, they used sodium N-lauroylsarcosinate (SAR)-PAGE for better differentiation of pegylated proteins, such as epoetin β pegol. Although WADA has recommended the use of pre-purification of samples, we developed a simple Western blotting method without pre-purification of samples. Instead of pre-purification, we used deglycosylation of samples before SDS-PAGE. The double detection of glycosylated and deglycosylated Epo bands increases the reliability of the detection of Epo protein. All of the endogenous Epo and exogenous ESAs shift to 22 kDa, except for Peg-bound epoetin β pegol. All endogenous Epo and exogenous ESAs were detected as 22 kDa deglycosylated Epo by liquid chromatography/mass spectrum (LC/MS) analysis. The most important factor for the detection of Epo is the selection of the antibody against Epo. WADA recommended clone AE7A5, and we used sc-9620. Both antibodies are useful for the detection of Epo protein by Western blotting.
直到世界反兴奋剂机构（WADA）开发出一种方法，才能检测到促红细胞生成素（Epo）。WADA 推荐使用等电聚焦（IEF）-PAGE 的 Western blot 技术，以显示天然 Epo 和注射的红细胞生成刺激剂（ESA）出现在不同的 pH 区域。接下来，他们使用十二酰基肌氨酸钠盐（SAR）-PAGE 来更好地区分聚乙二醇化蛋白，如培哚普利 β 聚乙二醇。尽管 WADA 建议对样品进行预纯化，但我们开发了一种简单的 Western blot 方法，无需对样品进行预纯化。我们在 SDS-PAGE 之前对样品进行了去糖基化，而不是预纯化。糖基化和去糖基化 Epo 带的双重检测增加了 Epo 蛋白检测的可靠性。除了结合 Peg 的培哚普利 β 聚乙二醇之外，所有内源性 Epo 和外源性 ESAs 都转移到 22 kDa。所有内源性 Epo 和外源性 ESAs 都通过液相色谱/质谱（LC/MS）分析被检测为 22 kDa 的去糖基化 Epo。检测 Epo 的最重要因素是选择针对 Epo 的抗体。WADA 推荐克隆 AE7A5，我们使用 sc-9620。这两种抗体都可用于 Western blot 检测 Epo 蛋白。
