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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 It is highly unlike that blood can be withdrawn from the body, treated and stored without change in any protein.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.

Code: 08C19YP 

Living at altitude increases haemoglobin and haematocrit, hence altitude-training is popular among endurance athletes. Since increases in haematocrit can be attained using illicit means like blood-doping or erythropoietin (Epo) use and are potentially hazardous, high haematocrit levels are used to exclude athletes from competition, also without evidence of doping. While sea-level athletes can choose to train at altitude, for others living at altitude has been a way of life for generations (e.g. east-Africans). Recent approaches developed to distinguish the effects of altitude on haematological profiles from those of blood-doping are approximately 20-80% successful. When applying these approaches to the haematological profiles of elite athletes, we found indications of systematic blood-doping or cases of naturally elevated blood markers. There is therefore an urgent need for these methods to be revised to remove any possibility of athletes being incorrectly banned from competition or, conversely, avoiding sanction due to broad definition of legal limits. In this project we will investigate standard red cell indices and contrast these following Epo administration in athletes not involved in competition. Gene-expression profiles will be assessed using the very latest gene-microarray technology. These results will be used to formulate new methods with improved discriminatory power relative to current detection protocols and in doing so eliminate the possibility of naturally elevated blood markers due to athletes living and/or training at altitude and unidentified doping due to inadequate detection. The use of gene-arrays, validated in this context, may provide gene-expression profiles relevant to other illegitimate approaches to improving oxygen carriage that may have been, or will be in the future, devised.

Main findings: 

The use of recombinant human erythropoietin (rHuEpo) is prohibited by the World Anti-Doping Agency. An OMICS-based longitudinal screening approach has the potential to improve further the performance of current detection methods such as the Athlete Biological Passport. For this project, we successfully used gene expression profiling in whole blood to identify genes that are differentially regulated following rHuEpo administration in Caucasian trained males and Kenyan endurance runners living at sea-level and moderate altitude (~2150 m), respectively. Relative to baseline, the expression of hundreds of genes were found to be altered by rHuEpo. In particular, 15 transcripts were profoundly up-regulated during the 4 weeks of rHuEpo administration and subsequently down-regulated up to 4 weeks post administration in both groups. Importantly, the same pattern was observed in all subjects. Furthermore, 30 transcripts were already differentially expressed two days after the first injection and are therefore promising candidate genes to detect microdose rHuEpo doping. The functions of the discovered genes were mainly related to either the functional or structural properties of the erythrocyte or to the cell cycle and its regulation. In summary, this research project successfully identified the blood “molecular signature” of rHuEpo administration and provided a set of candidate genes with potential to be robust biomarkers of rHuEpo doping. These preliminary results provide the strongest evidence to date that OMICS technologies such as gene expression have the potential to substantially improve and add a new dimension to the current anti-doping methods such the Athlete Biological Passport for rHuEpo detection.

Code: 08B01RG 

Protein hormones represent an extremely challenging analytical problem in terms of anti-doping control. The main reasons are that most of these proteins are produced by humans and that the concentrations in body fluids, such as blood or urine, are very low. Whereas the second condition puts stringent demands on the analytical instrument in terms of sensitivity the first condition does similar to the scientist’s ingenuity in order to enable differentiation between like and non-like. Thus far, all protocols addressing protein hormone doping are based on immunological techniques only and suffer from the problems like the unknown specificity of the antibodies in the assays, the potential cross-reactivity under different conditions, and the fact that all sample handlings cannot be monitored and only an end-stage reading is provided. Still, regardless the analytical measurement immunoglobulins will be required to address specifically a particular category of proteins in complex mixtures.  
This project aims at the development of a single step purification of all protein hormones from plasma through a multi-antibody platform followed by in-situ solid-phase proteolysis and nano-LC chip mass spectrometric identification and quantification. Three different phases can be distinguished for each protein hormone:  A- the characterisation of antibodies addressing a particular protein (or category) by means of surface Plasmon resonance (SPR). From this study the best immunoglobulin (in terms of surface bound properties, specificity and cross reactivity, thermodynamic parameters of the interaction, compatibility in a mixed antibody setting) will be selected.
B- extrapolation of the SPR results to a LC-compatible immunoaffinity stationary phase. For this purpose monoliths functionalised to use similar immobilisation chemistry as in SPR will be employed. Again, first single antibody monoliths shall be characterised and subsequently multi antibody monoliths will be build to finally address all protein hormones in a single step.
C- Identification and quantification of the IAC purified material. This will be accomplished by -1- eluting the IAC captured material onto a protease-containing stationary phase with a short (min) stop-flow setting to allow proteolysis -2- elution of the generated peptides into a reversed phase column to allow conventional chromatography -3- identification of the peptides from each hormone. Quantification shall be achieved using diagnostic peptides with specific isotopic labelling that will be injected as internal standards in each analysis.
The outcome should be a “all-in-one” single injection system that reduces sample manipulation/loss and addresses all hormones employing the same sample, recovering the remainder of the sample for other analytical procedures.

Main findings: 

The project AntiProDo aimed at the development of a single instrumental set-up that included purification of four protein hormones from plasma through a multi-antibody platform followed by in-situ solid-phase proteolysis and nano-LC chip mass spectrometric identification and quantification. 
Through a meticulous characterisation of the binding characteristics of multiple antibodies, one was selected for each of the four initial target analytes: hGH, EPO, hCG and IGF-I. Subsequently, customised monolith solid supports in silica capillaries were produced and functionalised with the antibodies. The functional behaviour was verified. Simultaneously, the same capillary support was developed to house proteolytic enzymes and the activity, efficiency and durability established. At another front, the target proteins were submitted to mass spectrometric analysis to establish the proteotypic peptides to target in a final setting. Heavy isotope labelled peptides were produced as internal standards and the analytical method based on nano-LC ms designed and validated. Ultimately, all elements were hyphenated to demonstrate the proof of concept for this approach.  
Further optimisation of the individual steps, particularly at the reproducibility in the manufacture of the functionalised capillaries, is required before this approach can be taken further.

Code: 08E09WS

The aim of blood manipulation is to increase the total hemoglobin mass (tHb-mass), which is directly correlated to maximum aerobic power and hence performance. When using the current doping tests it is not yet possible to detect autologous blood transfusions or the application of all kinds of erythropoiesis boosting stimulants.  
To minimize these illegal practices we recommend monitoring tHb-mass of endurance athletes over time. If the individual profile deviates substantially from that expected, the athlete has to undergo further follow-up testing. Serial measurements of tHb-mass can also be used to demonstrate objectively that an athlete has or had not used blood doping practices.  
Practical experience demonstrates that the recently developed method (optimized CO-rebreathing method) is valid, very reproducible and suitable to measure routinely an athlete’s tHb-mass. The procedure takes about 10 minutes and requires only a few drops of capillary blood. The practicability and significance of the method was evaluated within the last two years (2006-2007) in a multicentre study financially supported by a WADA grant. The individual tHb-mass profiles of >200 elite athletes of different endurance disciplines were screened and the preliminary results indicated good stability of tHb-mass (~2-3% variation).  
Doping with blood or EPO increases tHb-mass by at least 10-15%, whereas our recently obtained data (n>200) provide strong evidence that endurance training at sea level has no or only small effects on tHb-mass. It therefore follows that tHb-mass could be used as a screening parameter for blood manipulations. However, we also observed in individual cases that special influences can affect tHb-mass markedly. Without further systematic investigation, these special circumstances potentially undermine the utility of tHb-mass test to detect blood manipulation. The purpose of this follow-up project, therefore, is to identify physiological and outside influences (possible confounding factors) which could alter the normal stability of tHb-mass in athletes. Special emphasis will be placed the influence on tHb-mass of injury and severe infections, iron deficiency and iron supplementation, the impact of heavy and long-lasting exercise, and on the development of tHb-mass in junior athletes until maturation. Knowledge and quantification of these potentially confounding factors is of high importance to interpret deviations of a normal tHb-mass profile. Additionally, markers of these diseases will be correlated to tHb-mass, which will further increase the specificity of the test. As Germany and Australia provide the advantage of a central sports medical health system, large groups of the above-mentioned athletes can be recruited for the planned measurements. The proposed project will quantify factors that potentially confound the normal stability of tHb-mass athletes; specifically the effects of injury, severe infection, and of intense/long-lasting exercise and maturation. The long-term aim of the research team is to include tHb-mass in the “athlete’s biological passport” and to define subject-based reference ranges with the recently developed probabilistic inference techniques. The results of this ‘confounding factors’ study and its markers are essential for the application of tHb-mass into a “biological passport”. 

Main Findings:

Hemoglobin mass is closely related to aerobic performance and the aim of all kinds of blood manipulation is to increase Hb-mass und thereby the oxygen transport capacity. It is, there-fore, desirable to use Hb-mass as a screening tool for blood manipulation. From anti-doping studies we know, that blood manipulation by erythropoiesis stimulating agents and blood transfusions exceed the normal oscillation of Hb-mass. The effects of confounding factors are, however, not sufficiently investigated and may interfere with interpreting the effects of doping. 
The aim of this project was, therefore, to detect and to judge confounding factors which may explain possible variations in Hb-mass by other reasons than doping. In this study we focused on the effects on maturation, illness and injury, iron deficiency and iron supplementation, training breaks, and altitude effects. Additionally, methodological aspects of the CO-rebreathing method were considered. In total, data sets from 1881 CO-rebreathing tests of 428 elite and recreational athletes were obtained by two research groups from Australia and Germany. 
All those studies which were parallel conducted by both research groups yielded almost identical results and the typical error of the method was between 1.1% and 1.7%, i.e., both facts proved the method to be very robust and reliable. The CO-rebreathing procedure can be performed by the athlete in sitting or supine position, but it is influenced by acute exercise or by preceding intensive bouts of exercise. The half-time of COHb can be remarkably reduced by exercise or by breathing of an O2-enriched gas mixture. 
Illness and injury lasting until 2 weeks do not have a pronounced effect on Hb-mass whereas prolonged health problems leading to >2 week training interruption in rare cases (~1%) may decrease Hb-mass in the same magnitude as a blood donation. Severe iron deficiency and iron supplementation of anemic subjects has pronounced effects on Hb-mass and has to be taken into consideration. 
Normal changes in training volume have almost no effects, while complete training interruptions for more than 2 weeks have to be considered. 
Altitude training markedly increases Hb-mass if the stay lasts longer than 2 weeks and elevated values may exist until 5 weeks after return from altitude. 
Training has no effect in young children until puberty, and the most sensitive erythropoietic phase is between the age of 16 and 21 years. In adults above the age of 21 years no more age-related training effect do exist. Overall, our data show that the impact of the confounding factors on Hb-mass can be quantified and their effects can be included into statistical models, i.e. Bayesian approaches, as they are already used for the athlete’s biological passport (ABP). From the scientific point of view, we, therefore, conclude that the biomarker “Hb-mass” should be included into the ABP.

Code: 08B05PP 

Transfusing red blood cells (RBCs) is the oldest form of blood doping and - as the very recent findings around the Spanish cycling team Liberty Seguros, the German Cycling Team Telecom and further personal confessions of athletes demonstrate – is still or again used widely in top level sports. Autologous blood transfusion – up to now – is not detectable. It requires that the blood is stored for a longer period prior to reinfusion. 
Whereas red blood cells were for a long time regarded as mere O2-transport vehicles, there is evidence from recent studies that erythrocytes also possess enzymes that actively synthesize nitric oxide (NOS) and that alterations of NOS activation (translocation and phosphorylation) may be an indicator for autologous erythrocyte manipulation. Furthermore, adhesion receptors like CD47 antigen are present on RBCs and undergo alterations during blood manipulation. 
RBC NOS and adhesion receptors are detectable with immunohistochemical methods. Both are also be detectable by means of fluorescence activated cell sorting (FACS) after adaptation of the method, guaranteeing analysis of big numbers of blood samples in a short time. 
Our research group has demonstrated that the procedure of blood sampling, storage, and re-infusion in patients with hip-endoprothetic surgery induce an increase in RBC NOS activity in the RBC concentrate as well as in the patient’s blood after re-infusion. We also found RBC NOS activation during cardiopulmonary bypass. Furthermore, a progressive decrease in adhesion receptors in stored RBC concentrates could be demonstrated. Altogether, these data indicate, that an autologous blood transfusion induces a longer lasting activation of RBCs NOS system as well as changes in adhesion receptor expression. This has a high chance to be used for the detection of autologous blood transfusions when misused in sports. The relevant diagnostic approaches 
(immunohistochemistry plus gray-scale analysis and fluorescence activated cell sorting (FACS)) to quantify NOS and adhesion receptors of RBCs, therefore, may provide a new effective strategy in the fight against autologous blood doping. 
We will investigate 24 healthy male and female physically active subjects prior to 500 ml blood donation, as well as prior to and several days after re-infusion of the autologous blood concentrate after storage of 2 to 4 weeks. RBC NOS activity and adhesion receptors will be analysed in venous blood samples by means of immunohistochemistry and FACS. Furthermore, changes in blood total haemoglobin mass (tHb) will be analysed several times from prior to the donation until 2 weeks after re-infusion by means of optimized CO re-breathing method, allowing a quantification of the percentage of the re-infused amount of blood. Aerobic performance (V’O2max) will be tested several times for the analysis of the physiological effects of the procedures.

Main Findings:

Blood donation, but not red-blood cell re-infusion activated red-blood cell nitric oxid synthase (NOS) system at serine 116 and serine 1177 in healthy, moderately trained subjects. Therefore, red-blood cell NOS activation as measured by serine 116 and/or serine 1177 phosphorylation does not seem to be a valuable parameter for the detection of autologous blood doping in sports.  
However, there seems to be a physiological correlation between the red-blood cell NOS phosphorylation at serine 116 and the number of reticulocytes in the blood which remains to be elucidated in further studies. 

Code: 08A02JP

Recombinant glycoproteins expressed in non-human cells and in particular EPO and NESP has shown to contain small amounts of N-glycolyl-neuraminic acid (Neu5Gc), a sialic acid for which humans are devoid of the suitable hydroxylase. The detection of such non-human component of EPO (or other proteins) will constitute an unequivocal evidence of their exogenous origin. As part of previous research projects funded by WADA, we showed the presence of such monosaccharide in rEPO and NESP and later developed a capillary hplc method with fluorescence detection for the determination of small amounts of Neu5Gc (LOD ca. 5 fmol). By using such method in combination with an immunoaffinity purification developed in parallel using microwell plates coated with an anti-EPO antibody we were also able to detect the presence of Neu5Gc in plasma samples of EPO users. This method is already at the cutting-edge of the current technology using fluorescence detection. Furthermore, according to anti-doping regulations, the use of mass spectrometry is preferred, or required whenever possible. With this precedent, the aim of the present project is to move on following the same strategy, taking profit of our long experience in the field, and develop a method with a better sensitivity and able to detect Neu5Gc using mass spectrometry as unequivocal identification. The method will use nanoLC (hplc-chip from Agilent technologies) as the optimum sensitivity set-up and triple quadrupole MS. The instrument to be used has as specifications the detection of a chromatographic peak of 0.8 fmol reserpine (S/N =20) under conventional LC conditions. Using the hplc-chip (nanoLC) conditions this sensitivity should be increased ca. 50-100 times, fully supporting the feasibility of the attempt. The low amounts of Neu5Gc expected in urine of EPO users (ca. ≥ 35 fM) will be detectable in 20 mL of urine or less. Although some publications suggest that Neu5Gc can be incorporated into different glycoproteins through its ingestion as part of the diet, our preliminary results suggest that once a sample is immunopurified, there are no traces of Neu5Gc in negative samples, thus making the method specific for the purified glycoprotein. Although we will apply the method for EPO, using already developed immunopurification strategies, the same approach can be applied to the detection of the same analyte in different recombinant glycoproteins and matrices provided the corresponding specific immunopurification is applied.

Main Findings:

The detection of recombinant erythropoietin and/or its analogues is a major anti-doping problem because of the difficulty to differentiate exogenous from endogenous and the very low concentrations found in biological fluids. Current methods are based on electrophoresis and an anti-body recognition, not selective to the recombinant origin. The development of a ChipLC-MS nano-technique allows the analysis of conventional sample volumes through their pre-concentration in a built-in nano pre-column plus the on-line analytical separation through valve switching. All in a plug and play chip that includes the nanospray needle to be connected with an MS/MS detector. The objective of the current project was the development of an ultra sensitive analytical procedure for the detection of N-glycolyl-neuraminic acid as the unequivocal proof of the exogenous origin of EPO. The ChipLC/MS/MS procedure developed allowed a significant increase of sensitivity with respect to conventional instruments with a final limit of detection of 50 fmol Neu5Gc on column (as the DMB derivative) which, thanks to the large volume injection corresponds to 100 fmol/sample. The method showed to be linear and with acceptable precision and accuracy. However the instability of the nanospray needle made it not applicable for routine analysis. Furthermore, the sensitivity achieved, although significantly increased with respect to other MS procedures, is not amenable to the analysis of real samples yet. Still a limit of detection between 20 and 50 times lower would be needed. It is to be expected that as instruments become more sensitive, a method for the analysis of Neu5Gc on its own or a Neu5Gc containing peptide would be feasible in the coming years. 

Code: 08B15RG

The detection of human growth hormone in body fluids (plasma or urine) is a cumbersome task for several reasons being the low concentrations and the molecular heterogeneity the most important ones. Thus, at present for reasons of sensitivity and specificity immunoglobulins are indispensable in any analytical approach and also for the detection of growth hormone abuse antibodies play an important role. It is known that the administration of growth hormone results in a down regulation of the bodies-own production and that during a certain time frame only the exogenous molecule will be found in circulation. As the pharmaceutical product consists in a single isoform (the endogenous material is composed of several isoforms) this difference is used for anti-doping purposes.  Currently, two strategies are being followed: -1- an approach in which the amount one isoform (22 kDa) is compared all other isoforms and -2- an approach in which the amount of one isoform (22 kDa) is compared to the amount of a second isoform (20 kDa). For the second approach only a few specific antibodies are known and available. In order to have a reliable test for anti-doping purposes at least two specific antibodies, with distinct epitopes, should be available. In the course of the development of antibodies to the 20 kDa isoform fourteen different clones were generated by the research team. One, #7-clone 1B3, has now been fully explored. This antibody has shown superior surface properties with respect to the other known anti-20 kDa mAbs. Now, within the framework of this project the other clones will be produced at larger scale and characterised for their binding properties. The most promising clones will be further developed with the aim of having at least two distinct anti-20 kDa antibodies. The characterisation and comparison of the generated antibodies will be done by means of surface plasmon resonance and employing the entire arsenal anti-GH antibodies, GH isoforms, proteolytic GH fragments, synthetic GH isoforms, growth hormone binding proteins, sandwich assays, etc. This approach has already been applied successfully in the characterisation of the antibodies employed in the differential immunoassay that addresses the ratio between “pit” and “rec” isoforms. 

Main Findings: 

Human growth hormone (GH) in humans is a family of different molecules that come from a single gene. The composition of the family members fluctuates very little both at the intra- and interindividual level. However, when the GH pharmaceutical is administered the composition, and thus the ratio between the family members, is altered, and this sets the basis of the so-called direct approaches. One is the established rec/pit differential immuno assay approach that is implemented in most WADA accredited laboratories. An alternative approach, based on the specific ratio between 22 and 20 kDa GH is in the final stage of development with a multi-lab validation study. The major difficulty of the latter approach is the fact that finding a pair of antibodies (for screening and confirmation) with a different binding epitope for 20 kDa is very difficult as this variant differs only from the 22 kDa in that the amino acid Phe31 is linked to Asn47 instead of Glu32 (splicing-out of AA32-46). In this study we have evaluated and characterised 14 new anti-20 kDa antibody clones. We have identified 2 (antibody #4 and antibody #7) with excellent surface properties that are highly specific for 20 kDa GH. However, all antibodies that displayed binding to 20 kDa after immobilisation appear to have the same epitope as sandwich type studies reveal no complementary binding. When we compared the two best performers with one other established ultra specific and good surface antibody from a different source we could establish that this antibody, and the two identified here (#4 and #7) do bind different epitopes. As such, the 22 vs 20 kDa approach to address GH doping may be set up in compliance with the requirements for screening and confirmation procedures.