Progress in the Detection of Erythropoietin in Blood, Urine, and Tissue
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- Yukiko Yasuoka
- Department of Physiology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara 252-0374, Japan
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- Yuichiro Izumi
- Department of Nephrology, Kumamoto University Graduate School of Medical Sciences, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan
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- Jeff M. Sands
- Renal Division, Department of Medicine, Emory University School of Medicine, 1639 Pierce Drive, WMB Room 3313, Atlanta, GA 30322, USA
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- Katsumasa Kawahara
- Department of Physiology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara 252-0374, Japan
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- Hiroshi Nonoguchi
- Division of Internal Medicine, Kitasato University Medical Center, 6-100 Arai, Kitamoto 364-8501, Japan
書誌事項
- 公開日
- 2023-05-30
- 資源種別
- journal article
- 権利情報
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- https://creativecommons.org/licenses/by/4.0/
- DOI
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- 10.3390/molecules28114446
- 公開者
- MDPI AG
説明
<jats:p>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.</jats:p>
収録刊行物
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- Molecules
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Molecules 28 (11), 4446-, 2023-05-30
MDPI AG
