Detection of activin receptor type IIA and IIB-Fc fusion proteins by automated capillary immunoassay.

Activin receptor type IIA and type IIB fusion protein have been designed to sequester circulating molecules of the transforming growth factor-ß (TGF-ß) superfamily and inactivate their actions. Members of this superfamily have been reported as essential regulators of erythropoiesis by triggering the formation of activated ternary complexes containing different combinations of type I and type II receptors, which can limit RBC production by accelerating erythroid differentiation and inhibiting erythroid progenitor expansion. The recent approval of Luspatercept for the treatment of anemia associated to transfusion-dependent MDS and Beta-thalassemia in afflicted patients means that it can now pose a real threat of being abused in sport for its ability to stimulate erythropoiesis. Several methods for the detection of these molecules in blood have been proposed for the purpose of sport antidoping control. Here we propose the detection of the ActRIIA-Fc and ActRIIB-Fc fusion proteins by automated capillary Western immunoassay (Simple Western). The use of these immunoassays for the detection of protein targets has become widespread in the recent years. The work presented here demonstrates that this methodology enables a versatile, rapid, and sensitive detection of activin ligand traps in blood samples: plasma, serum, or dried blood spots (DBS). Preliminary results indicate that detection in urine samples is also possible. The option to use different antibodies allows the possibility to use this method as an initial testing procedure as well as a confirmation procedure. Finally, results coming from an administration study confirm that the method is suitable for routine analysis.

Standardization of reticulocyte counts in the athlete biological passport.

INTRODUCTION: The percentage of circulating reticulocytes (RET%) is a useful marker of blood doping in the context of the Athlete Biological Passport (ABP). The viability of the ABP depends on the comparability of sample data obtained across multiple laboratories for a given athlete. With the recent introduction of a different technology for the measurement of reticulocytes, the goal of this study was to compare currently employed Sysmex XT/XE analyzers to the recently introduced Sysmex XN analyzer. METHODS: RET% differences were searched in two independent data sets, the first consisting of 95 369 RET% values coming from 29 laboratories located in five continents as part of routine testing for the ABP, the second from a targeted study involving 510 samples analyzed on both a Sysmex XT and XN analyzers by two different laboratories. RESULTS: A relatively small but significant bias of 0.27 ([0.22-0.35] 95% CI) for the first data set and 0.19% ([0.16-0.22] 95% CI) for the second data set was observed with Sysmex XN analyzers returning higher values than Sysmex XT/XE analyzers. This bias appears constant over most of the range of RET% measured in elite athletes. CONCLUSION: When RET% values are obtained for the same athlete with different technologies (XT/XE vs XN), an adjustment of RET% emanating from the XT/XE instruments through a decrease of 0.22% within the ABP calculated ranges appears to be sufficient to integrate the results from the two technologies.

Detection of erythropoiesis stimulating agents in urine samples using a capillary Western system.

The presence of erythropoiesis stimulating agents (ESAs) in the urine samples collected from athletes is detected using traditional Western blotting following either size-based separation (SDS/SAR-PAGE) or isoelectric focusing (IEF). Although there is an important testing effort, there is little doubt that ESAs are still abused in sports and that reducing the costs of the tests might increase the number of tests and improve deterrence. The capillary electrophoresis system developed by Protein Simple may be useful to this end. This platform is fully automated and could be easily implemented in anti-doping laboratories, which would contribute to the improvement of the overall assay performance and standardization of the method. Such an automated system could be of interest during major sports events, such as the Olympic Games, where a high number of samples needs to be analyzed in a short period of time. From the experiments conducted so far, we conclude that the technique is promising, with the sensitivity and reproducibility needed to screen ESAs in human urine samples.

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

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. Copyright © 2017 John Wiley & Sons, Ltd.

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

Recombinant erythropoietin (rhEPO) has been misused for over two decades by athletes, mainly but not only in endurance sports. A direct rhEPO detection method in urine by isoelectric focusing (IEF) was introduced in 2000, but the emergence of third-generation erythropoiesis-stimulating agents and so-called biosimilar rhEPOs, together with the sensitivity of human endogenous EPO (huEPO) pattern to enzymatic activities and its modification following short strenuous exercise, prompted the development of a complementary test based on SDS-PAGE analysis. While Mircera and NESP are easily detected with the existing IEF and SDS-PAGE methods, some samples containing both epoetin-α/ß and huEPO present profiles that are still difficult to interpret. As doping practices have moved to micro-dosing, these mixed patterns are more frequently observed. We investigated the impact of enzymatic desialylation on the urinary and serum EPO profiles obtained by SDS-PAGE with the aim of improving the separation of the bands in these mixed EPO populations. We observed that the removal with neuraminidase of the sialic acid moieties from the different EPOs studied reduced their apparent molecular weight (MW) and increased the migration distance between huEPO and rhEPO centroids, therefore eliminating the size overlaps between them and improving the detection of rhEPO.

Involvement of tyrosine phosphatase CD45 in apoptosis.

CD45 is a transmembrane molecule with phosphatase activity expressed in all nucleated haematopoietic cells and plays a major role in immune cells. It is a protein tyrosine phosphatase that is essential for antigen-receptor-mediated signal transduction by regulating Src family members that initiate TCR signaling. CD45 is being attributed a new emerging role as an apoptosis regulator. Cross-linking of the extracellular portion of the CD45 by monoclonal antibodies and by galectin-1, can induce apoptosis in T and B cells. Interestingly, this phosphatase has also been involved in nuclear apoptosis induced by mitochondrial perturbing agents. Furthermore, it is involved in apoptosis induced by HIV-1. CD45 defect is implicated in various diseases such as severe-combined immunodeficiency disease (SCID), acquired immunodeficiency syndrome (AIDS), lymphoma and multiple myelomas. The understanding of the mechanisms by which CD45 regulates apoptosis would be very useful in disease treatment.

Low LC-MS/MS detection of glycopeptides released from pmol levels of recombinant erythropoietin using nanoflow HPLC-chip electrospray ionization.

The test used by anti-doping laboratories to detect the misuse of recombinant erythropoietin (rhEPO) is based on its different migration pattern on isoelectric focusing (IEF) gel compared with the endogenous human erythropoietin (hEPO) that can possibly be explained by structural differences. While there is definitely a need to identify those differences by LC-MS/MS, the extensive characterization that was achieved for the rhEPO was never performed on human endogenous EPO because its standard is not available in sufficient amount. The goal of this study was to develop an analytical method to detect pmol amounts of N-linked and O-linked glycopeptides of the recombinant hormone as a model. Using a nanoflow HPLC-Chip electrospray ionization/ion trap mass spectrometer, the diagnostic ion at m/z 366 of oligosaccharides was monitored in the product ion spectra to identify the four theoretical glycosylation sites, Asn24, Asn38, Asn83 and Ser126, respectively, on glycopeptides 22-37, 38-55, 73-96 and 118-136. With 3 pmol of starting material applied on Chip, only the desialylated N-glycopeptides 22-37 and 38-55/38-43 could be observed, and of all the glycan isoforms, those with the smaller structures were predominantly detected. While the preservation of the sialic acid moieties decreased the detection of all the N-glycopeptides, it allowed a more extensive characterization of the O-linked glycopeptide 118-136. The technique described herein provides a mean to detect glycopeptides from commercially available pharmaceutical preparations of rhEPO with the sensitivity required to analyze pmol amounts of hEPO, which could ultimately lead to the identification of structural differences between the recombinant and the human forms of the hormone.

Involvement of CD45 in DNA fragmentation in apoptosis induced by mitochondrial perturbing agents.

CD45 is a type I transmembrane molecule with phosphatase activity which comprises up to 10% of the cell surface area in nucleated haematopoietic cells. We have previously demonstrated the absence of nuclear apoptosis in CD45-negative T cells after chemical-induced apoptosis. The aim of this study was to characterize the role of CD45 in nuclear apoptosis. In contrast to wild type CD45-positive T cells, the CD45-deficient T cell lines are resistant to the induction of DNA fragmentation and chromatin condensation following tributyltin (TBT) or H2O2 exposure, but not to cycloheximide-induced apoptosis. CD45 transfection in deficient cell lines led to the restoration of chromatin condensation and DNA fragmentation following TBT exposure. In both CD45-positive and negative T cell lines, TBT exposure mediates intracellular calcium mobilization, caspase-3 activation and DFF45 cleavage. Moreover, DNA fragmentation was also induced by TBT in cells deficient in expression of p56lck, ZAP-70 and SHP-1. Subcellular partitioning showed a decrease in nuclear localisation of caspase-3 and DFF40. Together, these results demonstrate for the first time, that CD45 expression plays a key role in internucleosomal DNA fragmentation and chromatin condensation processes during apoptosis. CD45 activity or its substrates' activity, appears to be located downstream of caspase-3 activation and plays a role in retention of DFF40 in the nucleus.