Evaluation of AMGEN clone 9G8A anti‐Epo antibody for application in doping control. Issue 11 (21st September 2016)

• Record Type: Journal Article
• Authors: Reichel, Christian; Benetka, Wolfgang; Lorenc, Barbara; Thevis, Mario
• Is Part Of: Drug testing and analysis. Volume 8:Issue 11/12(2016:Nov./Dec.)
• Page Start: 1131
• Page End: 1137
• Publication Date: 2016-09-21
• Subjects: Clone 9G8A anti‐EPO antibody -- clone AE7A5 anti‐EPO antibody -- erythropoietin -- SAR‐PAGE -- SDS‐PAGE -- isoelectric focusing -- IEF‐PAGE -- doping control; Drugs -- Analysis -- Periodicals; Drug testing -- Periodicals; Chemistry, Forensic -- Periodicals; 615.1901
• DOI: 10.1002/dta.2057
• Languages: English
• ISSNs: 1942-7603

Abstract

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, zinc­α2­glycoprotein, 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. Abstract : Five test proteins (hZAG, E. coli TrxR, hNSE, hNNE, S. cerevisiae enolase), which showed medium to strong non‐specific interactions with clone AE7A5 anti‐EPO antibody, were evaluated regarding their binding behaviour to the non‐commercial anti‐EPO antibody clone 9G8A. Contrary to AE7A5, clone 9G8A was developed using full length EPO with the correct N‐terminal sequence. The antibody showed neither on SDS‐, SAR‐, nor IEF‐PAGE cross‐reactions with the five proteins. However, the sensitivity was lower compared to clone AE7A5. By applying immunoaffinity purification before electrophoretic separation, the non‐specifically bound proteins were removed and thus did no longer interfere with correct identification of endogenous and recombinant erythropoietins by AE7A5.