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

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.

Multiplexed detection of agents affecting erythropoiesis (AAEs) and overall strategy for optimizing initial testing procedure

“…SDS‐PAGE and SAR‐PAGE electrophoresis were performed as described by Martin et al 2 and Reihlen et al, 3 respectively. Electrophoresis were conducted using precast 10% Bis‐Tris Midi gels and MOPS‐based running buffer, at a constant 200 V for 70 min.…”

“…Proteins were transferred onto PVDF membrane using CAPS buffer discontinuous system after SDS‐PAGE 2 and Towbin buffer discontinuous system for SAR‐PAGE (in‐house validated system). The membrane was blocked in 5% nonfat milk (NFM)‐PBS for 45 min at room temperature.…”

“…ERAs detection in urine/serum/plasma from doping controls mainly refer to the detection of the various generations of recombinant erythropoietins (rEPOs). [1][2][3] Over the years, inclusions of new ERAs, improvements and modifications of the techniques, and efforts to harmonize detection have resulted in several revisions of the EPO WADA Technical Document describing procedures and ERAs identification criterias. 4 As an alternative to ERAs to treat disorders of erythropoiesis, novel drugs acting on the later stages of erythropoiesis to promote erythrocytes maturation have recently been developed.…”

“…Detection of ERAs for antidoping purpose is performed with well‐established procedures including three main steps: concentration/immunopurification of the sample, electrophoretic separation (IEF‐, SDS‐, or SAR‐polyacrylamide gel electrophoresis) and specific immunodetection. ERAs detection in urine/serum/plasma from doping controls mainly refer to the detection of the various generations of recombinant erythropoietins (rEPOs) 1–3 . Over the years, inclusions of new ERAs, improvements and modifications of the techniques, and efforts to harmonize detection have resulted in several revisions of the EPO WADA Technical Document describing procedures and ERAs identification criterias 4…”

A fast screening method for the detection of CERA in dried blood spots

“…Use of erythropoietin receptor agonists (ERAs)) for performance‐enhancing purposes can be detected via direct and indirect methods 1 . Direct detection typically relies on separation techniques that depend on the isoelectric point (IEF method) or molecular weight (e.g., sodium N ‐lauroylsarcosinate polyacrylamide gel electrophoresis [SAR‐PAGE] or SDS‐PAGE) of the ESA 1,2 …”