Desialylation improves the detection of recombinant erythropoietins in urine samples analyzed by SDS‐PAGE

Desharnais, Philippe; Naud, Jean-François; Ayotte, Christiane

doi:10.1002/dta.1494

Cited by 10 publications

(17 citation statements)

References 29 publications

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“…It turns out that the nature of the glycosylation, extent of branching and level of sialylation modulate the activity of such cytokines over a range of function, by affecting its interaction with its cognate receptor, and also by altering the rate of clearance from the circulation. In passing, it is worth mentioning that differences in the sulfation and sialylation of the N-glycans expressed on endogenous versus exogenous erythropoietin are used by the Anti-Doping Agency to detect illicit administration, and has led to rescinding of many major sporting trophies (645)(646).…”

Section: Tuning a Range Of Functionmentioning

confidence: 99%

Biological roles of glycans

2016

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Simple and complex carbohydrates (glycans) have long been known to play major metabolic, structural and physical roles in biological systems. Targeted microbial binding to host glycans has also been studied for decades. But such biological roles can only explain some of the remarkable complexity and organismal diversity of glycans in nature. Reviewing the subject about two decades ago, one could find very few clear-cut instances of glycan-recognition-specific biological roles of glycans that were of intrinsic value to the organism expressing them. In striking contrast there is now a profusion of examples, such that this updated review cannot be comprehensive. Instead, a historical overview is presented, broad principles outlined and a few examples cited, representing diverse types of roles, mediated by various glycan classes, in different evolutionary lineages. What remains unchanged is the fact that while all theories regarding biological roles of glycans are supported by compelling evidence, exceptions to each can be found. In retrospect, this is not surprising. Complex and diverse glycans appear to be ubiquitous to all cells in nature, and essential to all life forms. Thus, >3 billion years of evolution consistently generated organisms that use these molecules for many key biological roles, even while sometimes coopting them for minor functions. In this respect, glycans are no different from other major macromolecular building blocks of life (nucleic acids, proteins and lipids), simply more rapidly evolving and complex. It is time for the diverse functional roles of glycans to be fully incorporated into the mainstream of biological sciences.

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Section: Tuning a Range Of Functionmentioning

confidence: 99%

Biological roles of glycans

2016

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“…We have previously reported that the desialyation of EPOs after IP followed by SDS‐PAGE analysis could help discriminating EPO profiles of endogenous origins from those excreted following the administration of micro‐doses of rhEPO . In order to test whether desialylation improve the detection of “biosimilars” with a lower apparent molecular weight, serum samples were left untreated (Figure A, lane 3) or spiked with 20 IU/L of Hemax (Figure A, lane 4) and Beijing 4 Rings (Figure A, lane 5), with 10 IU/L of EPOmax (Figure A, lane 6) and analyzed by SAR‐PAGE.…”

Section: Resultsmentioning

confidence: 99%

“…Thereafter, non‐specific isolation of EPO by perchloric acid precipitation or more targeted isolation methods by EPO monoliths and immunoaffinity well plates were published . Some groups also attempted to use double‐step IP for the analysis of blood EPO by SAR‐PAGE or SDS‐PAGE . These methods are however necessarily paired to IEF or double‐blotting SAR‐PAGE analysis since some amounts of protein remain after IP and may interfere on single‐blotting of SAR‐PAGE.…”

Section: Introductionmentioning

confidence: 99%

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

Desharnais

Naud

Ayotte

2017

Drug Testing and Analysis

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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.

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“…Consequently, improved separation is achieved on SDS/SAR-PAGE 40. With the exception of simple sialidase treatment,41 this observation has not been exploited in doping analysis so far (figure 7). …”

Section: Newer Developmentsmentioning

confidence: 99%

Detection of peptidic erythropoiesis-stimulating agents in sport

Reichel

2014

Br J Sports Med

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Detection methods for erythropoiesis-stimulating agents in sport can be classified into direct and indirect approaches. Direct methods comprise electrophoretic techniques (isoelectric focusing (IEF-), sodium-dodecylsulfate (SDS-), sarcosyl (SAR-) polyacrylamide gel-electrophoreses (-PAGE)), ELISAs and mass spectrometric methods. The haematological module of the Athlete Biological Passport is currently the only applied indirect approach. Newer developments include a mass spectrometric test for peginesatide, sequential exoglycosidase digestion of ertythropoietin (EPO) combined with electrophoresis (SDS/SAR-PAGE), a dipstick method (MAIIA), and a study on the differences in sialic acid O-acetylation of tryptic EPO O-glycopeptides. The focus of this article is on direct detection methods.

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Desialylation improves the detection of recombinant erythropoietins in urine samples analyzed by SDS‐PAGE

Cited by 10 publications

References 29 publications

Biological roles of glycans

Biological roles of glycans

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

Detection of peptidic erythropoiesis-stimulating agents in sport

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