Projets de recherche

Code: 08B11CR

The detection of doping with recombinant peptide and protein hormones (e.g. erythropoietin – Epo, human growth hormone - hGH) is one of the most challenging analytical problems in doping control. The WADA-accredited method for the detection of doping with recombinant human erythropoietins (rhEpo) is based on isoelectric focusing (IEF). Our laboratory and the anti-doping control laboratory of Montreal developed – independently of each other – an SDS-PAGE method which serves as an additional confirmation tool for the worldwide practiced Epo-IEF method. The advantages and additional benefits of this method were presented at various scientific meetings in 2007 and 2008 (e.g. the capability to distinguish between Epo-biosimilars and effort urines, no interference by active urines). By applying this strategy we were able to clearly and multiply demonstrate the abuse of Dynepo by athletes – which is difficult to uncover with the IEF-method alone.Howewer, both the SDS-PAGE method and the Epo-IEF method use urine as sample matrix. Unfortunately, one of the latest generation Epo-pharmaceuticals (MIRCERA, a PEGylated Epoetin beta) is hardly excreted in urine due to its prolonged serum half-life and molecular mass (ca. 60 kDa). An ELISA test will be offered by the manufacturer for quantifying MIRCERA in blood. The consequences will be that in the future THREE methods will have to be performed in order to unambiguously detect the misuse of recombinant eyrthropoietins and analogs, i.e. the Epo-IEF method, the SDS-PAGE method for additional evidence (e.g. Dynepo, effort urines, biosimilars), and the serum/plasma ELISA for detecting MIRCERA abuse. Two different matrices will have to be used: blood and urine. The aim of this project is to develop a method which is capable of detecting doping with all forms recombinant erythropoietins in blood and in a single experiment.

Main findings

Recombinant erythropoietins perform with different sensitivity on SDS-PAGE after Western blotting. While the sensitivity of the majority of epoetins (e.g. epoetins alfa, beta, delta, omega; darbepoetin alfa) is similar on SDS-PAGE, the sensitivity of MIRCERA (PEGylated epoetin beta) is drastically decreased. Redesigning SDS-PAGE by exchanging the SDS for SARCOSYL in the sample and running buffers specifically enhanced the sensitivity for MIRCERA. SARCOSYL, a methyl glycine-based anionic surfactant with slightly higher CMC but much lower aggregation number than SDS, is not capable of solubilizing PEGs under PAGE-conditions - regardless of their polymerization degree (PEGs 1500 to 35000 were tested). Instead, SARCOSYL is only binding to the protein-part of MIRCERA leading to a sharp band on SAR-PAGE. SDS, on the other hand, is binding to both the PEG- and protein-chains of MIRCERA, which leads to band broadening on SDS-PAGE. As a result, the monoclonal anti-EPO antibody (clone AE7A5) is no longer binding to the fully - i.e. PEG- and protein-chain -solubilized MIRCERA-molecules, but only to those molecules which contain only SDS bound to the protein-chain. Naturally, these molecules are located on top of the band, since their charge density is reduced and their migration behaviour decreased. Because these molecules resemble only a small fraction of the MIRCERA-molecules originally loaded on the gel, a decrease in sensitivity is observed. SARCOSYL, on the other hand, leads to a sharp MIRCERA-band, since no solubilization of PEG-chains occurs. Consequently, the antibody is able to bind to all MIRCERA-molecules and no loss in sensitivity is observed after Western blotting. Besides, SARCOSYL-PAGE detects non-PEGylated epoetins with the same sensitivity and resolution as SDS-PAGE. The applicability of SAR-PAGE for detecting MIRCERA, recombinant epoetins, and endogenous EPO in blood and with high sensitivity could be demonstrated by performing single dose excretion studies. Besides, SAR-PAGE is not restricted to electrophoretic separations using the BisTris buffer system -e.g. MOPS-chloride boundary- but is fully compatible with other discontinuous buffer systems, namely the standard Laemmli (glycine-chloride boundary) [1], Neville (borate-sulfate boundary) [2], and Allen-Moore (e.g. borate-citrate boundary) [3] stacking systems – also indicating that the net-charge of the SARCOSYL-protein (i.e. erythropoietin, MIRCERA) micelles is stable within the pH-range of ca 7-10. The developed method is suitable for blood and urine, is not prone to „active“ and „effort urines“, is highly sensitive (down to femtogram-level, i.e. ca 10 amol) and with enhanced sensitivity compared to the traditional SDS-PAGE method for MIRCERA. The criteria of positivity (qualitative criteria, relative mobility values) are simpler since only one band instead of a series of isoforms and their distribution has to be evaluated. Of special importance is the fact that only one matrix and only one method are necessary for the detection of doping with all forms of recombinant erythropoietins (one matrix – one method approach) – instead of currently 4 methods and two matrices. Also, the sensitivity of SAR-PAGE for MIRCERA is higher than the sensitivity of IEF-PAGE (this was independently shown by the anti-doping control laboratory in Lausanne) – which is especially important for the screening. Hence, we also recommend the usage of SARPAGE as a screening procedure, because otherwise cases of low dosed MIRCERA would be missed by the IEF-PAGE method (false negatives). And finally, the required sample volume for SAR-PAGE is very low: 200 μL of serum are sufficient for the detection of shEPO and all forms of recombinant EPO.

Dr. Elsa Clara CORBELLA, Universidad Nacional de Córdoba, ARGENTINA

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

Code: 08C06CM

AIMS/SIGNIFICANCE: -differentiate blood from transfused and non-transfused individuals. - Correlate changes in protein pattern to Hb, physical performance and VO2 max. Finding markers of autologous blood doping is important in order to maintain the fundamental aspect of sports: fair play. BACKGROUND/HYPOTHESIS: Physical performance can be enhaced by blood boosting. Doping using the hormone EPO and homologous blood (non-self) can today be detected while autologous (self) blood transfusion is undetectable. Red blood cells (RBC) can be stored for up to 5 weeks in +4C and for several years in -80C. It is highly unlike that blood can be withdrawn from the body, treated and stored without change in any protein. METHODS: By using proteomic methods, thousands of proteins can be separated and identified. In combination with multivariate statistical methods protein markers to detect autologous blood transfusion can be found. Separation of proteins is done by 2D DIGE and proteon identification done by mass spectrometry. Wr can quantitatively detect changes in protein patterns, thereby separate blood from soped and un-soped individuals. RESULTS: A 10% difference in protein abundance can be detected (95%Cl). Over 2300 proteins protein spots can be separated from 100 uL of RBC. Fresh blood was comparared with blood stored for 5 weeks in -80C. 48 proteins were altered, including enzymes (e.g catalase) stress (E.G hsp 71) and structural (e.g actin) proteins. Ongoing experiments have detected -80 proteins changed by storage in +4 C for 5 weeks. STUDY DESIGN: After blood donation (10 subjects) and storage for 4 week at 4C, RBC will be reinfused. Blood samples will be taken from the subjects sevral times before and after donation and reinfusion. Control samples will be taken from a matched groups. Haemoglobin, Physical performance and VO2 max will be measured on 7 occasions.  

Main Findings: 

The specific aims of this study were: I. Differentiate blood from transfused and non-transfused individuals.
II. Correlate changes in protein pattern to Hb, physical performance and VO2max.       We have investigated the possibilities to use proteomics as a tool to screen the human Red blood cell (RBC) membrane proteome for novel and unique biomarkers useful for development of future diagnostic point-of-care tests. A comparison between fresh and freeze-stored (-80° C) RBC’s were performed using the 2D DIGE technique. From findings in freeze-stored blood, 20 candidate proteins were identified. 
A blood transfusion study was subsequently performed where 10 subjects underwent an autologous blood transfusion (2 x 450 mL donated whole was blood and 2 x 300 mL washed RBC’s re-infused) after 16 week freeze-storage of the RBC’s. Blood samples were drawn at 13 time points for hematological and proteomic analyses and physical performance testing done 9 times. 
Forty eight hours after blood transfusion, Hb increased by 5%, physical performance 
(Running time to exhaustion) was increased by 15% and VO2max by 16%. Only a weak correlation (R2 = 0.33) was seen between Running time and VO2max. 
Blood samples taken from the subjects as well as from the transfusion bags were analyzed by proteomic and standard clinical methods. There is a clear separation of blood taken from a freeze stored bag and fresh venous blood. Different protein profiles between blood taken before and after a transfusion can be visualized. Some of these results were confirmed by Western blot. 
Because no method is today available to directly detect an autologous blood transfusion, we believe that our method under development will provide a solution in a near future, and the current work-plan is to have a prototype (alpha-version) ready for testing within 18-24 months, pending funding and the speed of technical advancements. 

Code: 08C20TF 

AIMS OF THE PROJECT. This goal of this project has been to compare global patterns of gene expression as described in disparate WADA-sponsored studies of the effects of doping agents of methods such as erythropoietin or hypoxia, growth factors such as human growth hormone and IGF-1, steroids and others. To achieve that goal, we have continued to refine the informatics infrastructure and have developed protocols for the application of computational methods for large-scale meta analysis of gene expression data sets from three separate and independent doping studies. We approached the directors of a number of WADA-sponsored studies to obtain data bases that could all be subjected to uniform analytical procedures to identify those presumably few common features that might constitute rigorous markers of exposure to doping manipulation. We received extensive data sets from two other WADA-supported investigators – James Rupert of the University of British Columbia and Dr. Tejvir Khurana of the University of Pennsylvania – and have identified preliminary candidate signatures for further validation and comparison with results of additional data sets to be included in future analyses.   

Main Findings: 

 We have successfully used the WADA Informatics facility to down-load and analyze several large transcriptomic datasets, including the one generated in our own laboratory for the IGF-1 study (Bhasker and Friedmann, 2008), as well as datasets rom the WADA-supported studies of James Rupert at the University of British Columbia and Dr. Tejvir Khurana of the University of Pennsylvania. The purpose of these preliminary studies has been to identify and solve the up-loading difficulties that outside users might encounter. The results of that exercise are presented in detail in the attached figures. Briefly, we have demonstrated that a comparison of studies using disparate methods of creating hypoxic conditions in mice reveal similar patterns of transcriptional dysregulation, despite many major differences in experimental design. These similarities include established categories of biological processes, molecular function and specific gene aberrations (slides 4-6) of Powerpoint summary. Those similarities may constitute the beginnings of a rudimentary molecular “signature” for metabolic and gene expression responses to hypoxia and/or to possibly related manipulations such as artificially augmented blood production in a sport setting (Slide 7). In contrast, a comparison of hypoxia conditions with the expected “negative control” effects of IGF-1 exposure of muscle stem cells reveals fewer transcriptional changes in common with the hypoxic conditions, as expected. We emphasize that these results require extensive validation and corroboration with other related and unrelated data sets from other WADA investigators. That will be the emphasis for future studies with this system.

Code: 08C04MM

Different analytical approaches can be foreseen for direct analysis and for the identification of a characteristic signature pattern following gene doping. The pilot project will evaluate the proof of principle of Affinity Based Biosensors (ABB) integrated with bioinformatics and biomolecular approaches for gene doping detection. Our challenge is to provide a total analytical process for the evaluation of the presence of the gene doping event. The heart of the project is a new multi-screening and real time bioanalytical protocol, based on an affinity sensing platform to be used both in direct and indirect based approaches for gene doping detection. We believe affinity-based biosensors (ABBs), flanking conventional and profiling methodologies, can contribute to gene doping detection as fast, low cost and easy to use instrumental approach. In this context, a flexible platform, consisting of a biochip coupled to a label free technology for simultaneous measurements in short time could represent an innovative approach for selectively detecting gene doping markers (direct approach) or secondary effects induced by gene transfer (indirect approach). The feasibility of this project is assured by the high interdisciplinarity of the partners of the proponent team which will contribute with their specific competences to the definition of a total analytical process using bioanalytical, bioinformatics, biomolecular and immunological competences. The pilot project outcome, will be transferred to the Italian reference anti-doping laboratory (Federazione Italiana Medico Sportiva, Roma), accredited by WADA.

Main Findings

The present project aimed to develop an innovative analytical approach or delivering sampling and analytical protocol to be applied to gene doping detection, eventually setting up a database. A new multi-screening and real time bioanalytical protocol, based on an affinity sensing platform for gene doping detection was developed using an integrated multidisciplinary approaches based on bioanalytical, bioinformatics, biomolecular and immunological competences. In particular, we developed a bioinformatics supported study for the identification of suitable markers for gene doping tracing. The initial purpose of the Gene Doping Detection Database (GDDDB) is to provide functionality for the design of primers on sequences that can be potentially used as Vectors during gene doping. Since it is currently the most commonly used gene therapy vector, the pilot study GDDDB contains Adenovirus sequences only. The database scheme is designed so that it can be interfaced by a biomart engine (www.biomart.org). Primer design is done using the primer-BLAST web service provides by NCBI (www.ncbi.nlm.nih.gov/tools/primer-blast/) which is a web-based graphical interface to the Primer3 and BLAST algorithms. Here we describe the entity relationship diagram of the database, and the show how it is adjusted to a data warehouse scheme as required for use by the biomart engine. The GDDDB can be accessed and tested via the GDD portal at http:/aragon.cl.cam.ac.uk/GDD/dbportal.html. Furthermore a simple discrete Bayesian analysis is done to calculate the posterior probabilities if gene doping. These results are shown and are given for each probe used in the developed assay. The conditional posterior probabilities are also shown, depending on whether a high or low affinity has been observed from samples. The project also developed an animal model (in vivo approach). The in vivo approach has first used transgenic mice to for the EGFP reporter gene to validate the molecular analysis of the marker in different tissue. Once the applicability of the develop method for the analysis of the selected marker has been proved in this first transgenic model, then a second model system based on the injection of the vector, containing the same reporter gene EGFP, in the tibialis anterior and of the femoral quadriceps muscles was developed. The sampling has been executed at different times after transfection and from different tissues: muscles, liver, spleen, kidney, lung, heart, right quadriceps (site of injection) and left quadriceps. Moreover body fluids (urine, blood, tears) have also been used to evaluate the presence oof viral vector signature. To trace the marker gene dedicated approaches have been developed and applied to these animal models. The EGFP expression has also monitored in different tissue of transgenic mice. In order to unambiguously detect the presence of recombinant vector, a protocol for construct-specific sequences was also developed. For this purpose new primers pairs were designed respectively on 3' promoter and 5' end of the EGFP sequences. Finally different region of the EGFP marker was amplified for tracing the marker in the mice after gene-doping event mimicking. The target sequences were found in all the sampled tissue. Surface plasmon Resonance imaging (SPRi)-based sensing for the detection of the gene-doping event was achieved. In particular Affinity Based Biosensors (ABB) have been developed. Both for DNA target sequence detection (DNA sensing) and antibody detection in human serum (immunosensor) are reported. Immobilization chemistries for molecular robe surface binding, analytical protocol were first optimized using standard solutions and further applied to complex samples (PCR mixture for DNA sensing - direct approach and serum for indirect approach) The analytical platform (biochip) allows simultaneous and real-time detection of sequences belonging to the vector.