title: 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.
authors.affiliation: Doping Control Laboratory, AIT Seibersdorf Laboratories, Seibersdorf, Austria.|European Monitoring Center for Emerging Doping Agents, Cologne, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Reichel
authors.affiliation: Doping Control Laboratory, AIT Seibersdorf Laboratories, Seibersdorf, Austria.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Benetka
authors.affiliation: Doping Control Laboratory, AIT Seibersdorf Laboratories, Seibersdorf, Austria.
authors.forename: Barbara
authors.initials: B
authors.lastname: Lorenc
authors.affiliation: Institute of Biochemistry / Center for Preventive Doping Research, German Sport University Cologne, Cologne, Germany.|European Monitoring Center for Emerging Doping Agents, Cologne, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D004300:Doping in Sports; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004921:Erythropoietin / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029589:Seminal Plasma Proteins / Q000737:chemistry* / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D015813:Substance Abuse Detection / Q000379:methods*; D000090203:Zn-Alpha-2-Glycoprotein
chemical_list: D000911:Antibodies, Monoclonal; C554270:EPO protein, human; D011994:Recombinant Proteins; D029589:Seminal Plasma Proteins; D000090203:Zn-Alpha-2-Glycoprotein; D004921:Erythropoietin; D012967:Sodium Dodecyl Sulfate
keywords: Clone 9G8A anti-EPO antibody; IEF-PAGE; SAR-PAGE; SDS-PAGE; clone AE7A5 anti-EPO antibody; doping control; erythropoietin; isoelectric focusing