Universität zu Köln

Reichel, Christian and Thevis, Mario (2012). Detection of EPO-Fc fusion protein in human blood: Screening and confirmation protocols for sports drug testing. Drug Test. Anal., 4 (11). S. 818 - 830. HOBOKEN: WILEY-BLACKWELL. ISSN 1942-7603

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Abstract

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 (C) 2012 John Wiley & Sons, Ltd.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Reichel, Christian UNSPECIFIED UNSPECIFIED UNSPECIFIED Thevis, Mario UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-479494
DOI:	10.1002/dta.1381
Journal or Publication Title:	Drug Test. Anal.
Volume:	4
Number:	11
Page Range:	S. 818 - 830
Date:	2012
Publisher:	WILEY-BLACKWELL
Place of Publication:	HOBOKEN
ISSN:	1942-7603
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language IMMUNOGLOBULIN TRANSPORT PATHWAY; HUMAN ERYTHROPOIETIN; DOPING CONTROL; MASS ACCURACY; RECOMBINANT; RECEPTOR; PAGE; PHARMACOKINETICS; EXPRESSION; RESOLUTION Multiple languages Biochemical Research Methods; Chemistry, Analytical; Pharmacology & Pharmacy Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/47949