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Detection of EPO‐Fc fusion protein in human blood: Screening and confirmation protocols for sports drug testing
Author(s) -
Reichel Christian,
Thevis Mario
Publication year - 2012
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.1381
Subject(s) - erythropoietin , recombinant dna , fusion protein , detection limit , chemistry , pharmacology , medicine , chromatography , biochemistry , gene
The neonatal Fc receptor (FcRn) has been under investigation for several years as a pharmaceutical drug target. Clinical studies have shown that fusion proteins consisting of human recombinant erythropoietin (rhEPO) and the Fc‐part of IgG can be transported after pulmonary administration via FcRn across the airway epithelium to the blood stream. So far, no clinically approved pharmaceutical formulation of EPO‐Fc is available. Since various forms of recombinant erythropoietins have been frequently misused by athletes as performance‐enhancing agents, EPO‐Fc might play a similar role in sports in the future. In order to investigate the detectability of EPO‐Fc in human blood, different strategies were tested and developed. Only two of them fulfilled the necessary requirements regarding sensitivity and specificity. A rapid protocol useful for screening purposes first enriches EPO‐Fc from human serum via high capacity protein A beads and subsequently detects EPO‐Fc in the eluate with a commercial EPO ELISA kit. The limit of detection (LOD) of the method is about 5 pg (45 amol) EPO‐Fc and is independent of the serum volume used. For screening and/or confirmation purposes a second protocol was evaluated, which consists of a fast EPO immunopurification step followed by sodium dodecyl sulfate or sarcosyl polyacrylamide gel electrophoresis (SDS‐PAGE, SAR‐PAGE) and Western double‐blotting with chemiluminescence detection – a method already established in routine EPO anti‐doping control. The latter strategy allows the detection of EPO‐Fc in serum together with all other recombinant erythropoietins and with an identical LOD (5 pg/45 amol) as for the rapid screening protocol. Copyright © 2012 John Wiley & Sons, Ltd.