Immunomagnetic beads‐based isolation of erythropoietins from urine and blood for sports anti‐doping control

Abstract

According to the World Anti-Doping Agency (WADA) technical document for erythropoiesis stimulating agents (ESA) analysis (TD2014EPO), double-blotting of serum/plasma samples is mandatory for all analysis by isoelectric focusing (IEF) and for the confirmation procedures (CP) performed by SDS-PAGE or SAR-PAGE. The goal is to prevent potential cross-reactions 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-coated immunomagnetic beads and biotinylated anti-EPO polyclonal antibodies. Here we report that this immunomagnetic bead-based purification allows the analysis of serum/plasma samples by single-blotting. Serum and plasma samples, either intact or spiked with different ESAs, were immunopurified and analyzed by single-blotting, after SAR-PAGE or IEF using a cross-reaction minimized secondary antibody coupled to HRP. The results show that when samples are immunopurified according to this strategy, there is no non-specific binding when single-blotting is performed after SAR-PAGE. 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-Mouse IgG-coated magnetic beads and anti-EPO mAb). The recovery of ESAs was shown to be significant for serum/plasma samples. Our results suggest that single-blotting could be performed on serum/plasma samples without non-specific interferences.

Annual banned‐substance review: Analytical approaches in human sports drug testing

“…Irrespective, the need for improved test methods concerning the misuse of EPO and its analogs in sport has resulted in different studies aimed at expanding and fine‐tuning current strategies as well as exploring the value of alternative matrices and biomarker approaches . Desharnais et al investigated the utility of immunopurifying serum/plasma and urine samples by means of streptavidin‐coated magnetic nanoparticles in combination with a polyclonal biotinylated anti‐EPO antibody, in order ensure appropriate qualities of electrophoretic analyses by omitting the necessity of time‐consuming double blotting . The recovery of EPO, NESP, EPO‐Fc, and CERA was found between 58 and 100% and serum LODs were between 4 and 8 pg/mL (NESP, EPO‐Fc, and CERA) and 3.1 mIU/mL (recombinant human EPO BRP).…”

A simple method to immunopurify erythropoiesis stimulating agents from urine, aiming to optimize erythropoietin screening by SAR‐PAGE

“…Ultrafiltration has also been applied in combination with other methods such as immunomagnetic beads (LifeSpan Biosciences and GE Healthcare) and enzyme‐linked immunosorbent assay (ELISA; Stemcell Technologies). While immunomagnetic beads have been studied for their use on urine samples, they were introduced as an immunopurification method for blood samples . These beads can be coated with an antibody that binds selectively to the EPO molecule and reduces the occurrence of nonspecific binding.…”

“…These beads can be coated with an antibody that binds selectively to the EPO molecule and reduces the occurrence of nonspecific binding. Desharnais et al tested various coating‐antibodies to determine the best for capturing EPO specifically.…”

Combined immuno‐purification and detection of recombinant erythropoietins and activin receptor type II‐Fc fusion proteins by isoelectric focusing for application in doping control

“…Among other new ESAs in development, sotatercept and luspatercept, two activin receptor type II‐Fc proteins (ActRII‐Fc) from Acceleron Pharma now in phase 2/3 clinical trials, belong to a different class. Methods of identification in blood (due to their high MW, Fc‐fusion protein passage in urine is unlikely) by SAR/SDS‐PAGE have been described, but their detection by IEF remains to be explored. Interestingly, ActRII‐Fc proteins act at a later stage than rEPOs on erythropoiesis, and co‐treatment with the two type of ESAs seems to produce a synergic effect .…”