Purification of erythropoietin from human plasma samples using an immunoaffinity well plate

Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
J MallorquíJose A Pascual

Abstract

A method is described to isolate human erythropoietin (hEPO) from plasma using an EPO-specific immunoaffinity micro well plate (IAP). The operating conditions of the method (binding, blocking and elution) were optimised to avoid isoform discrimination and cross-contamination with other glycoproteins. The overall hEPO recovery was ca. 56% and significant clean-up for plasmatic hEPO was achieved. Polyvinylpyrrolidone (PVP) was used as a blocking reagent and elution took place at pH 11.0. Under these conditions all isoforms from recombinant human EPOs (rhEPOs) and analogues were uniformly recovered guaranteeing lack of discrimination. The resulting procedure allowed isolating erythropoietin from plasma in conditions amenable to hEPO analysis by other techniques such as SDS-PAGE or IEF. Moreover, avoiding contamination with other glycosylated material allowed the identification in human plasma samples of the non-human N-glycolyl-neuraminic acid (Neu5Gc) using HPLC-FLD. Neu5Gc is present as 1-2% of the sialic acid content in rhEPO so this approach could be used to unequivocally detect abuse of rhEPOs or analogues as part of the doping control.

References

Oct 6, 2000·Methods : a Companion to Methods in Enzymology·J E Butler
Oct 8, 2004·Internal Medicine·Wolfgang Jelkmann
Jun 2, 2007·International Journal of Biological Macromolecules·Françoise LasneJacques de Ceaurriz
Feb 5, 2008·Haematologica·Joaquim MallorquíJose A Pascual
Sep 23, 2008·Analytical Biochemistry·Esther LlopJosé A Pascual
Sep 1, 2009·Journal of Pharmaceutical and Biomedical Analysis·J MallorquíJ A Pascual

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Citations

Jun 4, 2011·Analytical and Bioanalytical Chemistry·Christian Reichel
Oct 11, 2017·Drug Testing and Analysis·Philippe DesharnaisChristiane Ayotte
Apr 30, 2014·The Analyst·S DefausR Gutiérrez-Gallego
Jan 22, 2011·Drug Testing and Analysis·Mario ThevisWilhelm Schänzer

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