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.

Comparison and optimization of SAR‐PAGE tests for erythropoietins in doping analysis

“…For standard single blotting, the membrane was incubated in a BlotCycler as described by Reihlen and Reichel . West Femto substrate was used for chemiluminescence.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing

“…In consideration of the continuously increasing sensitivity of mass‐spectrometry‐based test methods for glycoproteins such as EPO, targeting modified peptidic sequences of these biosimilars could offer alternative approaches towards detecting the misuse of these agents. Reichel and co‐workers assessed the utility of an alternative anti‐EPO antibody referred to as clone 9G8A for routine doping control purposes . Incorporating the antibody into commonly employed isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE) test methods, the clone 9G8A proved fit‐for‐purpose and cross‐reactivities with 5 critical proteins were not observed; however, the antibody was found to allow for only 40–45% of the sensitivity normally accomplished with the anti‐EPO antibody clone AE7A5.…”