{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,8]],"date-time":"2026-03-08T23:04:58Z","timestamp":1773011098786,"version":"3.50.1"},"reference-count":26,"publisher":"Wiley","issue":"11-12","license":[{"start":{"date-parts":[[2017,11,13]],"date-time":"2017-11-13T00:00:00Z","timestamp":1510531200000},"content-version":"vor","delay-in-days":12,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Drug Testing and Analysis"],"published-print":{"date-parts":[[2017,11]]},"abstract":"<jats:p>According to the World Anti\u2010Doping Agency (WADA) technical document for erythropoiesis stimulating agents (ESA) analysis (TD2014EPO), double\u2010blotting of serum\/plasma samples is mandatory for all analysis by isoelectric focusing (IEF) and for the confirmation procedures (CP) performed by SDS\u2010PAGE or SAR\u2010PAGE. The goal is to prevent potential cross\u2010reactions of the secondary antibody with remaining proteins in the purified samples. To this end, we have developed an immunopurification method of ESA in serum\/plasma samples using a combination of streptavidin\u2010coated immunomagnetic beads and biotinylated anti\u2010EPO polyclonal antibodies. Here we report that this immunomagnetic bead\u2010based purification allows the analysis of serum\/plasma samples by single\u2010blotting. Serum and plasma samples, either intact or spiked with different ESAs, were immunopurified and analyzed by single\u2010blotting, after SAR\u2010PAGE or IEF using a cross\u2010reaction minimized secondary antibody coupled to HRP. The results show that when samples are immunopurified according to this strategy, there is no non\u2010specific binding when single\u2010blotting is performed after SAR\u2010PAGE. With IEF, we observe a faint smearing, however, in the pH gradient outside the ESA detection region. These interferences did not alter ESA profiles of spiked urinary samples or of samples received for routine testing. This approach was compared to the MAIIA monoliths purification or to the isolation of ESAs with other combinations of immunomagnetic reagents (ie, anti\u2010Mouse IgG\u2010coated magnetic beads and anti\u2010EPO mAb). The recovery of ESAs was shown to be significant for serum\/plasma samples. Our results suggest that single\u2010blotting could be performed on serum\/plasma samples without non\u2010specific interferences. Copyright \u00a9 2017 John Wiley &amp; Sons, Ltd.<\/jats:p>","DOI":"10.1002\/dta.2320","type":"journal-article","created":{"date-parts":[[2017,10,9]],"date-time":"2017-10-09T22:06:40Z","timestamp":1507586800000},"page":"1744-1752","source":"Crossref","is-referenced-by-count":18,"title":["Immunomagnetic beads\u2010based isolation of erythropoietins from urine and blood for sports anti\u2010doping control"],"prefix":"10.1002","volume":"9","author":[{"given":"Philippe","family":"Desharnais","sequence":"first","affiliation":[{"name":"INRS\u2010Institut Armand\u2010Frappier Laboratoire de contr\u00f4le du dopage  531 Boul. des Prairies Laval Qu\u00e9bec Canada H7V 1B7"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8667-111X","authenticated-orcid":false,"given":"Jean\u2010Francois","family":"Naud","sequence":"additional","affiliation":[{"name":"INRS\u2010Institut Armand\u2010Frappier Laboratoire de contr\u00f4le du dopage  531 Boul. des Prairies Laval Qu\u00e9bec Canada H7V 1B7"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christiane","family":"Ayotte","sequence":"additional","affiliation":[{"name":"INRS\u2010Institut Armand\u2010Frappier Laboratoire de contr\u00f4le du dopage  531 Boul. des Prairies Laval Qu\u00e9bec Canada H7V 1B7"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2017,11,13]]},"reference":[{"key":"e_1_2_5_2_1","unstructured":"World Anti\u2010Doping Agency.Prohibited List January2017.https:\/\/www.wada\u2010ama.org\/sites\/default\/files\/resources\/files\/2016\u201009\u201029_\u2010_wada_prohibited_list_2017_eng_final.pdf. Accessed June 28 2017."},{"key":"e_1_2_5_3_1","unstructured":"WADATechnical Document \u2013 TD2014 EPO. Harmonization of analysis and reporting of erythropoiesis stimulating agents (ESA) by electrophoretic techniques.https:\/\/www.wada\u2010ama.org\/sites\/default\/files\/resources\/files\/WADA\u2010TD2014EPO\u2010v1\u2010Harmonization\u2010of\u2010Analysis\u2010and\u2010Reporting\u2010of\u2010ESAs\u2010by\u2010Electrophoretic\u2010Techniques\u2010EN.pdf. Accessed June 28 2017."},{"key":"e_1_2_5_4_1","doi-asserted-by":"publisher","DOI":"10.1055\/s-2007-989369"},{"key":"e_1_2_5_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/dta.2004"},{"key":"e_1_2_5_6_1","doi-asserted-by":"publisher","DOI":"10.1002\/dta.1411"},{"key":"e_1_2_5_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/dta.1915"},{"key":"e_1_2_5_8_1","unstructured":"C.Reichel P.Reihlen.EPO Working Group. Technical Note on the performance of SDS\/SAR\u2010PAGE for the detection of EPO\u2010doping. Communication of WADA to accredited laboratories.2014."},{"key":"e_1_2_5_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2007.04.002"},{"key":"e_1_2_5_10_1","doi-asserted-by":"publisher","DOI":"10.1002\/dta.171"},{"key":"e_1_2_5_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2010.09.034"},{"key":"e_1_2_5_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jchromb.2010.06.025"},{"key":"e_1_2_5_13_1","doi-asserted-by":"crossref","first-page":"494","DOI":"10.1002\/dta.97","article-title":"Sarkosyl\u2010PAGE: a new method for the detection of MIRCERA\u2010 and EPO\u2010doping in blood","volume":"11","author":"Reichel C.","year":"2009","journal-title":"Drug Test. Anal."},{"key":"e_1_2_5_14_1","doi-asserted-by":"publisher","DOI":"10.1002\/dta.1494"},{"key":"e_1_2_5_15_1","unstructured":"2015Anti\u2010Doping Testing Figures. WADA.https:\/\/www.wada\u2010ama.org\/ sites\/default\/files\/resources\/files\/2015_wada_anti\u2010doping_testing_figures_report_0.pdf. Accessed June 28 2017."},{"key":"e_1_2_5_16_1","doi-asserted-by":"publisher","DOI":"10.1002\/dta.1579"},{"key":"e_1_2_5_17_1","unstructured":"ZhouX XuY ZhangL. et al.Follow up research of two rhEPO AAF cases \u2013 comparison of erythropoietin electrophoretic migration behavior between urine and blood. Manfred Donike Workshop 35th Cologne Workshop on Dope Analysis.2017. (Oral presentation)"},{"key":"e_1_2_5_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0003-2697(02)00407-4"},{"key":"e_1_2_5_19_1","first-page":"122","article-title":"Comparative study of the EPO\u2010immunoaffinity approaches used for anti\u2010doping control purposes","volume":"23","author":"Desharnais P.","year":"2015","journal-title":"Recent Adv Doping Anal"},{"key":"e_1_2_5_20_1","doi-asserted-by":"crossref","first-page":"1323","DOI":"10.1182\/blood.V76.7.1323.1323","article-title":"A specific in vitro bioassay for measuring erythropoietin levels in human serum and plasma","volume":"76","author":"Wognum A. 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