An optimized SDS-PAGE protocol with a new blotting system for the initial testing procedure of ESAs in doping control

An optimized SDS-PAGE protocol with a new blotting system for the initial testing procedure of ESAs in doping control / Laurent Martin, Jean-Antoine Martin, Michel Audran, Alexandre Marchand. - (Drug Testing and Analysis (2020) 2 December)

  • PMID: 33269539
  • DOI: 10.1002/dta.2601


Abstract

Recombinant erythropoietins (rEPOs) are still among the substances endurance athletes use for doping. Detection methods are based on an electrophoretic separation of the proteins followed by a western blot and immunodetection with specific anti-EPO antibodies. In addition to IEF-PAGE, the SDS-PAGE method has been used to differentiate endogenous EPO from rEPOs by their molecular weight (MW). However, to adapt to new generations of rEPOs exhibiting higher MW, which were not well detected after SDS-PAGE, sodium lauroyl sarcosinate (SAR) is now used instead of sodium dodecyl sulfate (SDS) for the initial EPO testing procedure on doping control samples. The SAR-PAGE method is nevertheless expensive as it requires frequent buffer preparations using highly purified sarkosyl powder. In addition, this reagent needs to be handled with care due to acute toxicity by inhalation. The aim of this work was to improve the SDS-PAGE method by increasing its sensitivity and transfer of high-MW rEPOs. First, using a biotinylated primary anti-EPO antibody and avoiding the use of a secondary antibody increased the general sensitivity of both SDS-PAGE and SAR-PAGE to all rEPOs about four-fold. Then, by changing the buffer system during the protein transfer, with a CAPS buffer and a discontinuous buffer transfer system, high-MW rEPOs, EPO-Fc and CERA were transferred with higher efficiency and detected with high sensitivity. This optimized SDS-PAGE protocol could be adopted by anti-doping laboratories as an alternative to SAR-PAGE.

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Science
Study
Date
2 December 2020
People
Audran, Michel
Marchand, Alexandre
Martin, Jean-Antoine
Martin, Laurent
Country
France
Language
English
Laboratories
Paris, France: Agence Française de Lutte contre le Dopage (AFLD)
Analytical aspects
Testing method development
Doping classes
S2. Peptide Hormones, Growth Factors
Substances
Erythropoietin (EPO)
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22 April 2021
Date of last modification
4 May 2021
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