Universität zu Köln

Reihlen, Phillipp, Blobel, Mike, Weiss, Patrick, Harth, Judith, Wittmann, Julia, Leenders, Frank and Thevis, Mario . Introduction of a PEGylated EPO conjugate as internal standard for EPO analysis in doping controls. Drug Test. Anal.. HOBOKEN: WILEY. ISSN 1942-7611

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Abstract

Immunopurification of doping control samples is a mandatory necessity in erythropoietin (EPO) analysis during a confirmation procedure; moreover, it has become common practice to also immunopurify samples for the initial testing procedure. Typically used materials (e.g., Stemcell purification plate and MAIIA purification kit) rely on anti-EPO antibodies for purification. Also, the detection of EPO after electrophoretic separation and western blotting is based on a monoclonal anti-EPO antibody, clone AE7A5, directed against a 26 amino acid sequence of the N-terminal region of human EPO. While the electrophoretic separation and blot transfer efficiency can be monitored with reference standards and quality control samples, it is presently not possible to monitor the functionality of the entire sample preparation procedure. The reliance on antibodies for both purification and detection has complicated the implementation of an internal standard (ISTD). In this study, customized EPO-polyethylene glycol (PEG) conjugates were synthesized as potential ISTDs and assessed as to their compatibility with existing sample preparation procedures for urine and blood sample analysis using the most common immunopurification techniques. Moreover, probing for the impact of the ISTD on sodium N-lauroylsarcosinate (sarcosyl) polyacrylamide gel electrophoresis (SAR-PAGE)-based EPO analysis concerning potential interference with target analytes was conducted. The presented data demonstrate that a 12-kDa PEG residue attached to human EPO represents a particularly useful construct to serve as ISTD for erythropoietin-receptor agonist (ERA) analysis. The conjugate is applicable to both urine and blood testing using the commonly employed purification techniques, supporting and improving result interpretations especially concerning specimens where the natural abundance of human EPO is low.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Reihlen, Phillipp UNSPECIFIED UNSPECIFIED UNSPECIFIED Blobel, Mike UNSPECIFIED UNSPECIFIED UNSPECIFIED Weiss, Patrick UNSPECIFIED UNSPECIFIED UNSPECIFIED Harth, Judith UNSPECIFIED UNSPECIFIED UNSPECIFIED Wittmann, Julia UNSPECIFIED UNSPECIFIED UNSPECIFIED Leenders, Frank UNSPECIFIED UNSPECIFIED UNSPECIFIED Thevis, Mario UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-573054
DOI:	10.1002/dta.3211
Journal or Publication Title:	Drug Test. Anal.
Publisher:	WILEY
Place of Publication:	HOBOKEN
ISSN:	1942-7611
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language CHROMATOGRAPHY-MASS SPECTROMETRY; RECOMBINANT; ERYTHROPOIETIN; PROTEIN; PAGE Multiple languages Biochemical Research Methods; Chemistry, Analytical; Pharmacology & Pharmacy Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/57305