Evaluation of AMGEN clone 9G8A anti-Epo antibody for application in doping control
- PMID: 27552163
- DOI: 10.1002/dta.2057
Evaluation of AMGEN clone 9G8A anti-Epo antibody for application in doping control
Abstract
The two mouse monoclonal anti-erythropoietin (EPO) antibodies clone AE7A5 (generated by using a 26 amino acid N-terminal EPO-peptide) and 9G8A (developed by immunizing mice with full length human EPO) are both directed against linear epitopes at the N-terminus of EPO. While AE7A5 has been commercially available for many years, 9G8A was made for Amgen's internal research purposes. In the past, the commercial antibody was shown to cross-react with several proteins unrelated to EPO (e.g. E. coli thioredoxin reductase, zinc-α2-glycoprotein, S. cerevisiae enolase, human neuron-specific enolase, and human non-neuronal enolase). However, it displayed high sensitivity for detecting recombinant EPO (rEPO) misuse by athletes on Western blots. We evaluated the potential use of clone 9G8A for doping control purposes. While 9G8A showed lower sensitivity than AE7A5 (ca 45% on isoelectric focusing (IEF)-polyacrylamide gel electrophoresis (PAGE), ca 40% on sodium dodecyl sulfate (SDS)- and sarcosyl (SAR)-PAGE), non-specific binding of the five proteins was not observed. The cross-reactivity of AE7A5 can be overcome by immunoaffinity purification of EPO before electrophoresis and Western blotting. Similar to AE7A5, clone 9G8A is also suited for Western double-blotting. Copyright © 2016 John Wiley & Sons, Ltd.
Keywords: Clone 9G8A anti-EPO antibody; IEF-PAGE; SAR-PAGE; SDS-PAGE; clone AE7A5 anti-EPO antibody; doping control; erythropoietin; isoelectric focusing.
Copyright © 2016 John Wiley & Sons, Ltd.
Similar articles
-
Identification of zinc-alpha-2-glycoprotein binding to clone AE7A5 antihuman EPO antibody by means of nano-HPLC and high-resolution high-mass accuracy ESI-MS/MS.J Mass Spectrom. 2008 Jul;43(7):916-23. doi: 10.1002/jms.1444. J Mass Spectrom. 2008. PMID: 18576436
-
Data from a microdosed recombinant human erythropoietin administration study applying the new biotinylated clone AE7A5 antibody and a further optimized sarcosyl polyacrylamide gel electrophoresis protocol.Drug Test Anal. 2023 Feb;15(2):163-172. doi: 10.1002/dta.2989. Epub 2021 Jan 21. Drug Test Anal. 2023. PMID: 33450134
-
SARCOSYL-PAGE: a new method for the detection of MIRCERA- and EPO-doping in blood.Drug Test Anal. 2009 Nov;1(11-12):494-504. doi: 10.1002/dta.97. Drug Test Anal. 2009. PMID: 20355164
-
Progress in the Detection of Erythropoietin in Blood, Urine, and Tissue.Molecules. 2023 May 30;28(11):4446. doi: 10.3390/molecules28114446. Molecules. 2023. PMID: 37298922 Free PMC article. Review.
-
Erythropoietin and erythropoiesis stimulating agents.Drug Test Anal. 2012 Nov;4(11):805-12. doi: 10.1002/dta.1341. Epub 2012 Apr 16. Drug Test Anal. 2012. PMID: 22508651 Review.
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials
Miscellaneous