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Evaluation of AMGEN clone 9G8A anti‐Epo antibody for application in doping control
Author(s) -
Reichel Christian,
Benetka Wolfgang,
Lorenc Barbara,
Thevis Mario
Publication year - 2016
Publication title -
drug testing and analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.065
H-Index - 54
eISSN - 1942-7611
pISSN - 1942-7603
DOI - 10.1002/dta.2057
Subject(s) - enolase , blot , microbiology and biotechnology , monoclonal antibody , antibody , clone (java method) , epitope , polyacrylamide gel electrophoresis , gel electrophoresis , chemistry , recombinant dna , erythropoietin , isoelectric focusing , biochemistry , biology , enzyme , dna , immunology , immunohistochemistry , gene , endocrinology
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.