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Combined immuno‐purification and detection of recombinant erythropoietins and activin receptor type II‐Fc fusion proteins by isoelectric focusing for application in doping control
Author(s) -
Martin Laurent,
Audran Michel,
Marchand Alexandre
Publication year - 2019
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.2476
Subject(s) - isoelectric focusing , recombinant dna , activin receptor , fusion protein , phlebotomy , chromatography , erythropoietin , erythropoietin receptor , chemistry , receptor , medicine , biochemistry , gene , enzyme
Iso‐electric focusing (IEF) was the first method established to discriminate endogenous and recombinant erythropoietins (rEPOs). It is still approved by the World Anti‐Doping Agency (WADA) as an initial testing procedure to detect erythropoiesis stimulating agents (ESAs) in doping control samples. However EPO‐Fc, one of the prohibited rEPOs designated by WADA, is not detectable with the actual IEF conditions. Other newly developed ESAs – luspatercept and sotatercept, both activin receptor type II‐Fc fusion proteins (ActRII‐Fc) – are also now prohibited and could be used in combination with rEPOs. Methods of identification of ActRII‐Fc in blood by SAR/SDS‐PAGE have been described, but not by IEF. Here we detail improvements in blood sample preparation and IEF analysis: A combined immuno‐purification of EPOs and ActRII‐Fc proteins in a single procedure, an appropriate isoforms separation for all proteins using new pre‐loading and gel conditions, and a single detection of all rEPOs and ActRII‐Fc proteins after successive incubation with anti‐EPO and anti‐ActRII antibodies. With these changes, distinctive profiles for all the ESAs were obtained by IEF. Therefore, IEF could be used as a screening method to detect a wide spectrum of prohibited ESAs in blood samples prior to specific confirmation for the identified rEPO or ActRII‐Fc.