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Author Notes:

Hiroshi Nonoguchi: nono@insti.kitasato-u.ac.jp; Tel.: +81-48-593-1212

Y.Y., Y.I. and H.N. designed the research; Y.Y. and H.N. prepared the figures; Y.Y., Y.I., K.K. and H.N. wrote the manuscript. J.M.S. checked the manuscript. All authors have read and agreed to the published version of the manuscript.

We thank Drug Testing and Analysis (Wiley) for giving us permission to reuse Figure 1, Figure 2 and Figure 3 (License Agreement with Copyright Clearance Center 1355326, 1354320, and 1353916, respectively). We thank Elsevier for giving us permission to use the published figure in Heliyon (Ref. [79]). We also thank Physiological Reports (Wiley, Periodicals on behalf of The Physiological Society and the American Physiological Society), Molecules (MDPI), and J Biol. Chem (Elsevier) for the use of the figures under the Creative Commons Attribution License. Figure 10 is the summary of the methods described in this paper.

The authors have no financial conflict to declare.

Subjects:

Research Funding:

This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan 18K08247 (YI) and by the Science Research Promotion Fund from Kitasato University Medical Center (H24-011) (HN).

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Physical Sciences
  • Biochemistry & Molecular Biology
  • Chemistry, Multidisciplinary
  • Chemistry
  • erythropoietin
  • glycoprotein
  • deglycosylation
  • Western blotting
  • doping
  • HIF2 alpha
  • PHD inhibitor
  • hypoxia
  • HYDROXYLASE INHIBITOR ROXADUSTAT
  • RECOMBINANT-HUMAN-ERYTHROPOIETIN
  • CHRONIC KIDNEY-DISEASE
  • HEPATITIS-C
  • BIOLOGICAL-ACTIVITY
  • DARBEPOETIN-ALPHA
  • SUGAR CHAINS
  • LC-MS/MS
  • ANEMIA
  • HYPOXIA

Progress in the Detection of Erythropoietin in Blood, Urine, and Tissue

Tools:

Journal Title:

MOLECULES

Volume:

Volume 28, Number 11

Publisher:

Type of Work:

Article | Final Publisher PDF

Abstract:

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.

Copyright information:

© 2023 by the authors.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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