A simple method to immunopurify erthyropoiesis stimulating agents from urine, aiming to optimize erythropoietin screening by SAR-PAGE.
agent, doping
erythropoiesis stimulating
erythropoietin
immunopurification
Journal
Drug testing and analysis
ISSN: 1942-7611
Titre abrégé: Drug Test Anal
Pays: England
ID NLM: 101483449
Informations de publication
Date de publication:
Nov 2019
Nov 2019
Historique:
received:
11
08
2019
revised:
26
10
2019
accepted:
29
10
2019
pubmed:
7
11
2019
medline:
13
5
2020
entrez:
7
11
2019
Statut:
ppublish
Résumé
The efficiency of the immunopurification step of urinary erythropoietin (EPO) and recombinant forms is important for their optimal detection in antidoping screening. Previous investigations of immunopurification techniques have been done for immunomagnetic beads, EPO Purification Kit (EPK) columns (MAIIA Diagnostics), and enzyme-linked immunosorbent assay (ELISA) microplates (Stemcell Technologies) conjugated/coated with anti-EPO antibodies. In this study, a new immunopurification technique using anti-EPO sepharose gel beads, developed by MAIIA Diagnostics, to simplify and minimize sample handling was evaluated. This EPO Purification Gel Kit (EPGK) was compared with our current routine EPK for limit of detection (LOD). Linearity, recovery, repeatability, sample incubation time, and sample volume were also evaluated for EPGK. The LODs and linearity for EPK and EPGK were comparable to each other and the recovery for BRP, NESP, CERA, and EPO-Fc were within the range of other studies, and concentration of the sample eluate improved the recovery results. Little variation was seen within days, between days, and between operators. A 90 minute incubation of the sample with the sepharose gel beads is sufficient for most of the erythropoiesis stimulating agents (ESAs) tested, with 10 mL being an optimal sample volume for EPGK. The improved sample handling, higher sample throughput and the reduced working time demonstrate that the EPGK is a better alternative to the current MAIIA EPK immunopurification method for urine. The EPO Purification Gel Kit (from MAIIA Diagnostics) was evaluated and validated for immunopurification of endogenous erythropoietin and exogenous erythropoiesis stimulating agents from urine samples. The kit was a better alternative to that currently used (EPO Purification Kit) in many antidoping laboratories because it improves sample handling and increases sample throughput.
Substances chimiques
EPO protein, human
0
Hematinics
0
Erythropoietin
11096-26-7
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1666-1674Informations de copyright
© 2019 John Wiley & Sons, Ltd.
Références
Lasne F, Martin L, Crepin N, de Ceaurriz J. Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones. Anal Biochem. 2002;311(2):119-126.
Reichel C, Kulovics R, Jordan V, Watzinger M, Geisendorfer T. SDS-PAGE of recombinant and endogenous erythropoietins: benefits and limitations of the method for application in doping control. Drug Test Anal. 2009;1(1):43-50.
Reichel C, Gmeiner G, Reihlen P, et al. SARCOSYL-PAGE: optimized protocols for the separation and immunological detection of PEGylated proteins. Methods Mol Biol. 1855;2019:131-149.
Reihlen P, Blobel M, Kempkes R, et al. Optimizing SAR-PAGE. Drug Test Anal. 2015;7(11-12):1014-1016.
World Anti-Doping Agency. WADA Technical Document: Harmonization of analysis and reporting of erythropoiesis stimulating agents (ESAs) by electrophoretic techniques. https://www.wada-ama.org/sites/default/files/resources/files/WADA-TD2014EPO-v1-Harmonization-of-Analysis-and-Reporting-of-ESAs-by-Electrophoretic-Techniques-EN.pdf2014 [cited 29 July 2019].
Lasne F. Double-blotting: a solution to the problem of non-specific binding of secondary antibodies in immunoblotting procedures. J Immunol Methods. 2001;253(1-2):125-131.
Masquelier M, Heiland C, Bhuiyan H, et al. Optimization of ESAs detection in urine and blood: comparison of different immunopurification techniques. Recent Advances in Doping Analysis: 36th Cologne Workshop on Doping Analysis; 2018; Cologne, Germany.
Desharnais P, Naud JF, Ayotte C. Immunomagnetic beads-based isolation of erythropoietins from urine and blood for sports anti-doping control. Drug Test Anal. 2017;9(11-12):1744-1752.
Marchand A, Buisson C, Martin L, Martin JA, Molina A, Ressiot D. Report on an anti-doping operation in Guadeloupe: high number of positive cases and inferences about doping habits. Drug Test Anal. 2017;9(11-12):1753-1761.
Reihlen P, Völker-Schänzer E, Majer B, et al. Easy-to-use IEF compatible immunoaffinity purification of erythropoietin from urine retentates. Drug Test Anal. 2012;4(11):813-817.
Lönnberg M, Dehnes Y, Drevin M, et al. Rapid affinity purification of erythropoietin from biological samples using disposable monoliths. J Chromatogr A. 2010;1217(45):7031-7037.
Kwan SH, Baharudin F, Salim SA, et al. Evaluation of methods for purifying erythropoietin from urine using immunoaffinity applications. Int J Biol Chem. 2015;9(3):123-132.
MAIIA Diagnostics. EPO Purification Gel Kit - For Urine http://media.maiiadiagnostics.com/2019/06/DfU-101350-04-EPO-Purification-Gel-Kit-for-Urine.pdf [cited 29 July 2019].
MAIIA Diagnostics. EPO Purification Kit http://media.maiiadiagnostics.com/2018/06/Directions-for-Use-EPO-Purification-Kit.pdf2016 [updated 2018; cited 29 July 2019].
Reichel C. The overlooked difference between human endogenous and recombinant erythropoietins and its implication for sports drug testing and pharmaceutical drug design. Drug Test Anal. 2011;3(11-12):883-891.
World Anti-Doping Agency. International Standard for Laboratories https://www.wada-ama.org/sites/default/files/resources/files/isl_june_2016.pdf2016 [cited 7 August 2019].
MAIIA Diagnostics. EPO Purification Kit - For Blood http://media.maiiadiagnostics.com/2019/05/Direction-for-use-EPO-Purification-Gel-Kit-for-Blood.pdf [cited 2019 July 29].