Recent developments in doping testing for erythropoietin
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The constant development of new erythropoiesis-stimulating agents (ESAs), since the first introduction of recombinant erythropoietin (rhEpo) for clinical use, has also necessitated constant development of methods for detecting the abuse of these substances. Doping with ESAs is prohibited according to the World Anti-Doping Code and its prohibited list of substances and methods. Since the first publication of a direct and urine-based detection method in 2000, which uses changes in the Epo isoform profile as detected by isoelectric focusing in polyacrylamide slab gels (IEF-PAGE), the method has been constantly adapted to the appearance of new ESAs (e.g., Dynepo, Mircera). Blood had to be introduced as an additional matrix, because Mircera (a PEGylated Epo) is best confirmed in serum or plasma after immunoaffinity purification. A Mircera ELISA was developed for fast screening of sera. With the appearance of Dynepo and copy epoetins, the additional application of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE or equivalent) became necessary. The haematological module of the Athlete Biological Passport is the latest development in multivariable indirect testing for ESA doping. The article summarizes the main strategies currently used in Epo anti-doping testing with special focus on new developments made between 2009 and 2010.
KeywordsErythropoietin (Epo) Doping control Electrophoresis Mass spectrometry ELISA Biological passport Direct and indirect detection
In 1985 Lin et al.  described the cloning of the human Epo gene, which was the basis for the expression of the gene in transfected cell lines. Epo is a glycoprotein hormone produced mainly by the kidneys, and is responsible for the development of bone marrow stem cells into red blood cells (RBC, erythrocytes), which transport oxygen to organs and tissues . The entire process which leads to the production of RBC is called erythropoiesis, and substances which stimulate RBC production are called erythropoiesis-stimulating agents (ESAs). An increase in RBC is performance enhancing, especially in endurance sports (e.g., cycling, running, cross-country skiing). The first recombinant human Epo (rhEpo; epoetin alfa; Eprex, Erypo, Procrit) was approved in 1989 for clinical use. Because misuse by athletes was suspected, the International Olympic Committee (IOC) put Epo on its list of prohibited substances in 1990 . However, no IOC-approved test existed at that time. In 2000 a direct test was published by Lasne et al.  together with a blood-property-based indirect test . The direct test became the first WADA-approved method for Epo-doping control and was based on the principle that rhEpo application may lead to a temporary change in the endogenous Epo isoform distribution. This observation was made as early as the 1990s by other scientists (see the section “IEF-PAGE”). Further ESAs followed in 1990 (epoetin beta; NeoRecormon), 2001 (darbepoetin alfa; Aranesp or NESP), 2002/2007 (approval/launch of epoetin delta; Dynepo), and 2007 (methoxy polyethylene glycol–epoetin beta; Mircera, continuous erythropoietin receptor activator, or CERA). After the IOC, the World Anti-Doping Agency (WADA; established in 1999) became responsible for the prohibited list. The 2011 version of the list  bans ESA doping according to article S2 “Peptide hormones, growth factors and related substances” (prohibited substances) and also M2 “Gene doping” (prohibited methods). The misuse of non-approved substances is covered by article S0 “Non-approved substances”. The detection of Epo-doping has been regulated by WADA in a technical document (TD2009EPO) , and comprises mostly gel electrophoretic methods (i.e. isoelectric focusing (IEF), sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) or equivalent). Additional methods have been developed, including enzyme-linked immunoassays (ELISA), membrane-assisted isoform immunoassays (MAIIA), mass spectrometry-based methods, and procedures for the detection of gene doping and sample manipulation with proteases. The purpose of the “Athlete Biological Passport” and its “haematological module” is to enable prolonged detection of Epo doping by statistical application of indirect (blood profile) markers in a multivariable, longitudinal, and athlete-specific. Currently, the following techniques are used by WADA-accredited laboratories for detection of ESA doping: gel electrophoretic methods (IEF-PAGE, SDS-PAGE or equivalent), ELISA-based methods (measurement of Epo and Mircera concentrations), protease tests, and the determination of haematological data (“haematological module” consisting of haematocrit (HCT), haemoglobin (HGB), red blood cells count (RBC), percentage of reticulocytes (RET%), reticulocytes count (RET#), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and index of stimulation (OFF-hr score)).
GEL Electrophoretic methods
Methods based on the electrophoretic separation of Epo molecules and subsequent immunological detection (Western blot) are still dominant in detection of Epo doping. They are direct detection methods similar to immunoassay-based methods, but additionally allow a visualization of the antibody-bound molecules regarding charge heterogeneity (IEF-PAGE) or molecular mass (SDS-PAGE, SAR-PAGE). Based on this information a differentiation between recombinant and endogenous erythropoietins is possible.
A brief description of the method used in doping control
human urinary Epo contains greater amounts of acidic isoforms than rhEpo and shEpo; and
this difference can be used to detect the administration of rhEpo in both serum and urine .
Key improvements by Lasne et al.—namely the use of PAGE and the development of the so-called Western double-blotting technique—led to a test which, since then, has been widely used for the detection of Epo doping. In its original form the test uses urine (typically 20 mL), which is concentrated after adjustment with 3.75 mol L−1 Tris-HCl buffer to pH 7.4, sedimentation, and microfiltration in two ultrafiltration steps (30 kDa molecular weight cut-off) down to ca 20–40 μL. A mix of protease inhibitors is added during the entire sample-preparation procedure. Adjustment of the pH of the sample is necessary because it keeps (intact) Epo isoforms negatively charged (their isoelectric points (pI) are below pH 7.4); this prevents the action of acidic proteases and glycanases (e.g. neuraminidases) contained in urine, and also helps to dissolve urinary precipitates which may contain Epo. The retentate obtained after ultrafiltration contains Epo but also a large amount of urinary proteins (e.g. 20–40 μg μL−1). To avoid overloading effects (band deformation, smearing) during the subsequent electrophoretic separation, Epo can be affinity purified (e.g. by wheat germ agglutinin (WGA)  or anti-Epo antibodies [12, 13, 14]) before IEF-PAGE, the concentration of Epo in the retentate determined by ELISA and adjusted, e.g. to 500 IU L−1, for each sample, or methyl red (a pH indicator with a pI of 3.8) can be added to the IEF catholyte for accurate band identification. Typically, 5% T (total acrylamide) and 3% C (bisacrylamide crosslinker) polyacrylamide gels are used which contain carrier ampholytes in the pH 2–6 range at a final concentration of 2% (w/v), 7 mol L−1 urea, and 5% sucrose for stabilizing the pH gradient. After a prefocusing step to establish the pH gradient, retentates are heated for 3 min at 80 °C, supplemented with 1% Tween-80 or Tween-20, and applied cathodically to the gel by use of sample application pieces, a sample applicator strip, or—preferably—precast wells in the gel (vide infra). The main focusing step is performed at constant wattage (1 W cm−1 gel length and a 1 mm thick gel) and for 3600–4000 V h. Proteins are then semi-dry electroblotted on to a polyvinylidene fluoride (PVDF) membrane and incubated after a reduction (5 mmol L−1 dithiothreitol (DTT) in phosphate-buffered saline, PBS) and a blocking step (5% non-fat milk (NFM) in PBS) with the primary antibody (mouse monoclonal anti-Epo antibody clone AE7A5 in 1% NFM in PBS). The membrane is then washed with 0.5% NFM in PBS and the bound antibody transferred under acidic semi-dry blotting conditions (0.7% acetic acid) to a second PVDF membrane. Lasne [15, 16] introduced this procedure to avoid interfering interactions of the secondary antibody (usually a polyclonal goat anti-mouse IgG antibody) with urinary proteins other than Epo on the first membrane (Note: a similar technique using also a “donor” and “acceptor” membrane for the detection of antibody cross-reacting proteins was published as early as 1992 by Hammerl et al.  and is known as “Western cross blot”). The successful introduction of this so-called “double-blotting” technique was a key step in the development of the WADA-approved Epo IEF-PAGE method for doping-control purposes. The second membrane is then treated as a regular Western blot, i.e. blocked (5% NFM in PBS), incubated with a secondary biotinylated antibody, washed (0.5% NFM in PBS), treated with streptavidin horseradish peroxidase (HRP) complex, washed again (PBS), and developed using an enhanced chemiluminescence substrate. A CCD camera is used as signal detector, which enables accurate quantification of the bands obtained within each Epo isoform profile. The IEF profiles are evaluated after quantitative software-based image analysis [18, 19, 20] and in accordance with the criteria of the WADA technical document on Epo analysis (TD2009EPO) .
The WADA technical document TD2009EPO
Recent research results
Zinc alpha-2-glycoprotein (ZAG) was identified in 2008  as a protein which interacts non-specifically with the primary anti-Epo antibody used during the IEF procedure (clone AE7A5). Because ZAG focuses outside the region of the IEF gel which is used to evaluate the IEF profiles, no interference with the final results occur. The binding was known for several years [12, 29, 30] but the protein was not identified until 2008. A shotgun proteomics approach enabled the successful identification. Confirmation experiments using the Western double-blotting procedure and a recombinant version of ZAG showed that the interaction occurs only at high concentrations of ZAG (i.e. μg range on the gel). Earlier observations of non-specific binding of clone AE7A5 antibody made in the context of two-dimensional electrophoresis [31, 32] or SDS-PAGE [32, 33, 34] could either not be confirmed for the one-dimensional carrier ampholyte-based IEF-PAGE method or were shown to occur outside the pI range used for Epo testing [35, 36, 37, 38]. An additional and mandatory immunoaffinity purification step was introduced for SDS-PAGE-based analyses with TD2009EPO .
Lamon et al. (2009)  performed a study to identify conditions which lead to so-called “effort (atypical) profiles” and “effort urine samples”. Top cyclists had to perform a series of short strenuous exercises (e.g. four 500-m rides at maximum speed), and urine and blood were collected before and after the exercises. After the exercise a ca 300-fold increase in retinol binding protein (RBP) was observed in the urine and 8.5 and 10-fold increases in total protein and Epo, respectively. IEF-PAGE was performed after immunoaffinity purification  of urine and serum samples. The authors clearly demonstrated that this type of exercise led to “effort profiles” in urine but to no changes of the shEpo IEF profile. The data were explained by a possible “exercise-induced transient renal dysfunction” , which resulted in the excretion of more basic (less acidic) Epo isoforms . As was shown by Lasne et al. in 2007, shEPO is less acidic than uhEpo  and this may be used to explain the shift.
Discriminant analysis—a supervised multivariate statistical analysis technique frequently used in chemometrics—was applied by Lasne et al. (2007)  and Lamon et al. (2010)  for classification of IEF profiles. It could be demonstrated that this technique enabled improved detection of low-dose (microdose) rhEpo application [41, 43] and is also applicable to the detection of Dynepo doping . Lamon et al. proposed use of a so-called “bands intensity (BI) score” as alternative criterion or for additional evidence in the detection procedure of Epo doping by the IEF method.
Because of the low excretion in urine, Mircera should preferably be detected in blood. Usually, serum or plasma samples have to be immunoaffinity purified to remove high-abundance proteins, which would otherwise distort the IEF profile [12, 27]. Lasne et al. (2010)  developed a fast precipitation procedure which kept shEpo, rhEpos, NESP, and Mircera in solution and thus enabled direct application of the supernatant to the IEF gel after washing and ultrafiltration.
The effect of heparin on the detection of Epo by IEF-PAGE was investigated by Reichel et al. (2010) . To reduce the risk of life-threatening thromboembolic events (stroke, heart-attack) some athletes use anticoagulants in combination with Epo . Heparin is a highly sulfated glycosaminoglycan and well known anticoagulant. Because of its polyanionic nature, unfractionated heparin (UFH) acts destructively in IEF-PAGE. Concentrations of ca 25 IE UFH or above led to a complete distortion of the Epo profile. No such effect was observable for low-molecular-weight heparins (LMWH), which completely migrated to the anode. UFH on the other hand generated a heparin gradient across the entire pH 2–6 range of the gel and thus disturbed the separation of the Epo isoforms. Immunoaffinity purification of EPO before IEF-PAGE or treatment of the urinary retentate with solid urea removed the effect of UFH. No such interference of UFH was observed in the separation of Epo on SDS-PAGE.
Sample application pieces have frequently been used to apply urinary retentates to IEF gels. In 2007 interference was observed which affected the separation of NESP isoforms and which could be ascribed to faulty application pieces. Reichel (2010)  performed a mass spectrometric study on working and defective application pieces and showed that defective application pieces (bought between 2007 and 2010) contained a complex mixture of alcohol ethoxylates, alcohol ethoxysulfates, and alkyl sulfates including SDS. Anionic detergents like these are, in general, incompatible with IEF-PAGE. Casting IEF gels with wells and directly applying the retentates into the wells was demonstrated to be the best solution to this problem.
Kohler et al. (2008)  used two internal standards with masses above and below epoetins alfa, beta, delta, and uhEpo, which enabled the calculation of relative mobility values and subsequent classification of these analytes in samples. Reichel et al. (2009)  showed that effort urine samples and unstable (“active”) urine samples can be also easily detected by SDS-PAGE without the need to perform a stability test. Contrary to IEF-PAGE the degradation of Epo in active urine samples causes a shift of the uhEpo band to lower molecular mass (i.e. toward the anode) whereas the IEF profile is shifted toward the cathode. The position of the Epo band of effort urine samples is hardly altered compared with the band of the uhEpo standard and with no shifts to the rhEpo region. These data were confirmed by Voss et al. (2010), who investigated the effect of high-intensity exercise on the relative mobility values of Epo . Since 2009, SDS-PAGE has also been part of the WADA technical document on Epo analysis (TD2009EPO; vide supra).
IEF-PAGE for detection of epoetins alfa, beta, NESP, and some biosimilar compounds in urine;
SDS-PAGE for detection of Dynepo in urine;
IEF-PAGE for detection of Mircera in serum and/or plasma; and
Mircera-ELISA for screening of serum samples before confirmation with IEF-PAGE.
it is not a high-throughput method (the capacity is only one sample per gel);
very acidic epoetins, for example NESP, are only poorly resolved on commercial IPG strips, which end at pH 3 in the acidic region (although Görg et al. (2009)  demonstrated that good resolution of NESP is possible on non-commercial IPG strips which started at pH 2.5); and
it requires that all isoforms of the different epoetins migrate reproducibly and equally well from the first dimension IPG strip into the second dimension SDS-PAGE gel (otherwise artificial changes in the isoform distribution might occur).
enzymatically pretreated (NESP), or
pretreated with a precipitant (Mircera) before the application of the ELISA, OR
the elution characteristics of the ELISA’s capturing antibodies were used after sample incubation.
A fourth principle, use of species-specific differences in the primary structure of Epo, has been used for detection of human recombinant Epo in non-human samples (vide infra).
In an attempt to generate anti-NESP antibodies Giménez et al. (2007)  raised polyclonal anti-sera against two NESP peptides, LVNSSQVNETLQLHVC and QVNETLQLHVDKAVSGLRSC (amino acids 81–95 and 86–104), and one Epo-peptide, LVNSSQPWEPLQLHVC (amino acids 81–95). The main idea was to exploit differences between the primary structures of rhEpo and NESP to generate NESP-specific antibodies (a total of five amino acids were exchanged in the amino acid sequence of Epo for NESP, namely the amino acids in positions 30 (A→N), 32 (H→T), 87 (P→V), 88 (W→N), and 90 (P→T)). A specific interaction for NESP was observed for the anti-NESP (84–106) antibody but only after immunoaffinity purification using a NESP (86–91) peptide column. No cross-reaction with NESP was observed for the Epo (81–95) antibody. Nevertheless, despite their potential the specific application of these antibodies for anti-doping-control purposes has not been reported.
The different amino acid sequences of human Epo, NESP, and horse Epo were exploited by Lasne et al. (2005)  for detection of horse doping with rhEpo and NESP. An ELISA from R&D Systems was used for the screening procedure. Because antibodies of the ELISA had only weak cross-reactivity with equine Epo, a single dose injection of 200 μg NESP led to a significant increase in measurable Epo in plasma during the first 24 h after the injection (156 IU L−1). After ten days the basal value (<2.5 IU L−1) was reached again. A similar but weaker reaction was also observed after treatment with epoetin alfa. However, after the last injection the concentration in plasma rapidly decreased and reached the starting value within 48–72 h. The results were confirmed by use of the IEF method with horse urine as sample matrix. A similar approach was described by Bartlett et al. (2006)  for the application of ELISA kits in canine doping control. Dogs received 2500 IU of epoetin alfa (subcutaneously and intravenously) and urine samples were taken before and after administration of the drug. Three human Epo ELISA kits were tested (R&D Systems, Neogen, Immulite). The R&D systems kit gave the highest Epo values. Typically, the maximum concentration was observed 3.75 h after administration (e.g. 179 mIU mL−1), which dropped below 0.2 mIU mL−1 after 32.3–49.3 h. Again, the ELISA kits were used for screening purposes only, and confirmation was done with the IEF method. The method was tested with ca 6000 greyhound sport samples and resulted in four positive cases—three for rhEpo and one for uhEpo (because of submission of human urine instead of greyhound urine to the control stewards). However, it must be pointed out that administration of rhEpo to horses and dogs may lead to the production of Epo antibodies, because of the different amino acid sequences of human, equine, and canine Epo, and, consequently, harmful immunological reactions after repeated use of rhEpo .
A screening method for detection of Mircera in serum using a commercial Epo ELISA assay (Access Epo assay; Beckman Coulter) in combination with fractionated protein precipitation was published by Van Maerken et al. (2010) . The method is based on the observation that PEGylation of rhEpo (i.e. Mircera) leads to an increase in water solubility and that this increased solubility can be used for specific enrichment of Mircera contained in serum by precipitation of serum proteins with PEG-6000 . A final PEG-6000 concentration of 25% (w/v) was obtained by mixing 150 μL serum with 150 μL PEG-6000 (50% (w/v); dissolved in 0.15 mol L−1 saline solution). After vortex mixing and incubation at 37 °C (15 min) the samples were vortex mixed again and centrifuged (9300 g for 10 min). The supernatant was collected, diluted with 0.15 mol L−1 saline solution (1:4), and the Epo concentration was measured by use of the Access Epo assay—a magnetic bead-based immunoassay with chemiluminescent detection. Data were normalized by calculating the ratio between the Epo concentration obtained with and without PEG-6000 pretreatment (in the latter case 150 μL 0.15 mol L−1 saline solution was used instead of the 50% PEG-6000 solution). The authors observed a significant increase in the PEG:control ratio for haemodialysis patients (average value 2.15) treated intravenously with Mircera, compared with untreated patients (average value 0.92 for untreated non-renal patients and 0.82 for untreated haemodialysis patients). Additional longitudinal studies showed that the test was able to detect Mircera for two weeks in all the Mircera-treated patients (receiving 50–350 μg Mircera every four weeks) and in most cases during the third and fourth weeks also. It was concluded that the assay might also be useful for screening purposes in doping control, despite the fact it was developed using samples from clinically ill persons.
Lamon et al. (2009)  described the development and validation of an ELISA for detection of Mircera doping in human serum. The assay used a streptavidin-precoated microtitre plate for immobilization of a biotinylated polyclonal anti-Epo capture antibody. The coating procedure was preferably performed overnight. After a washing step 100 μL sample was added to the ELISA well and incubated for 2 h at room temperature. The well was washed again and then the bound Mircera was detected by a monoclonal anti-PEG antibody, which was labelled with digoxigenin (DIG; incubation time 1 h). To detect the bound anti-PEG antibody another incubation step was necessary—this time with an anti-DIG-Fab-HRP conjugate and a subsequent colorimetric reaction (2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] diammonium salt (ABTS) substrate solution; measurement of absorption at 405 nm, with 490 nm as reference wavelength). The lower limit of quantification (LLOQ) of the assay was 30 pg mL−1 and the lower limit of quality control (LLQC) 50 pg mL−1. For determining the cut-off limit of the ELISA (100 pg mL−1) 140 blank serum samples and serum samples from six Mircera administration studies were analysed and an ROC curve calculated. On the basis of this cut-off value the specificity and sensitivity of the assay were 100% and 80%, respectively, for a period of four weeks and a single injection of 200 μg Mircera. Depending on the route of application (intravenous, subcutaneous) and interindividual variability, detection windows between eight days and more than four weeks were obtained. Currently, the test is used by several doping-control laboratories for screening purposes and in combination with the IEF method for confirmation. The ELISA is commercially available from MicroCoat Biotechnologie (Germany). However, alternative strategies include the combination of IEF+IEF-PAGE, Sarcosyl-PAGE  +IEF-PAGE, and SDS-PAGE+IEF-PAGE.
A competitive ELISA for the detection of Hematide in plasma samples from test animals (rat, monkey) was published by Fan et al. (Affymax) in 2006 . Sample dilutions were first pre-incubated with a fixed amount of a polyclonal anti-Hematide rabbit antibody and then transferred to microtitre wells, which were coated with Hematide. Only antibodies which were not blocked by the Hematide in the sample were still able to bind to the Hematide on the wells. After a washing step the assay was developed by using an anti-rabbit IgG conjugate. The quantification limit of the ELISA was 31.5 ng mL−1 and it detected both the free and the PEGylated peptides. However, it was not reported whether this assay might also be useful for detection of Hematide in human samples.
Membrane-assisted isoform immunoassay (MAIIA)
However, the Epo contained in biological samples (e.g. serum, urine) must first be immunoaffinity purified before it can be applied to the EPO WGA MAIIA test strip. For this purpose Lönnberg et al. developed disposable monolith columns (6 μL, 0.15 mm length), which enabled rapid Epo purification . Approximately 20–40 μg of a mouse monoclonal anti-Epo antibody (clone 3 F6) was immobilized on each column. Bound Epo was desorbed under acidic conditions and in a small volume (ca 50 μL) of buffer. Average recoveries of endogenous Epo in urine were ca 65%. Recovery depended on the type of Epo and was lowest for Mircera (ca 30% in urine). The columns were also tested for a possible change of the Epo IEF profile after immunoaffinity purification, but no alteration was observed . The applicability of membrane-assisted isoform immunoassay as a screening procedure in Epo-doping control is currently in the validation phase.
Detection of proteases
The observation that Epo profiles of some urine samples were unstable led to the speculation that enzymes contained in these urine samples might be the reason. Hence, a stability test has to be routinely performed on all Epo IEF-PAGE confirmation analyses (vide supra). A small aliquot of the urine (500 μL) is supplemented with a mixture of protease inhibitors including pepstatin-A (an inhibitor of acid proteases), buffer-exchanged to pH 5.5 with acetate buffer, and spiked with rhEpo and NESP . Any shifts in the Epo profile are indicative of an unstable (“active”) urine (vide supra). The nature of this instability was ascribed to the presence of, e.g., proteases, glycosidases, sulfatases, or neuraminidases (the last are very active under the pH conditions of the test) because of bacterial proliferation during transport of the samples [40, 90]. To inhibit the growth of microorganisms, freezing of urine samples immediately after collection  or addition of 1% sodium azide  was therefore recommended. High enzymatic activity also led to completely empty IEF profiles in the pH range 2–6, because Epo degradation products had higher pI values. The effect of successive neuraminidase degradation on the IEF profile of rhEpo, which finally leads to asialoerythropoietin, was shown by Imai et al. as early as 1990 . Degradation by neuraminidases also leads to a decrease in molecular mass of Epo, which was demonstrated by Yanagawa et al., by SDS-PAGE, in 1984 . Belalcazar et al.  performed controlled digestions of uhEpo, rhEpo, and NESP with neuraminidase and arylsulfatase, and obtained profiles similar to those observed for unstable urine samples. Anielski et al. (2009)  showed, by urine spiking experiments, that bacteria causing urinary tract infections (E. coli, K. pneumoniae) were able to alter Epo IEF profiles in a manner similar to that observed for unstable urine samples.
However, undetectable Epo IEF profiles might also be caused by the intentional addition of enzymes to the urine during sample collection by the athlete. Thevis et al. (2007) developed a procedure which enabled detection of such manipulation . A threshold of 15 μg mL−1 protease concentration (measured with a protease activity test) was established, above which further investigations should be made. The threshold was based on the observation that in most tested urine samples endogenous protease activity was below 6 μg mL−1 and that at 20 μg mL−1 (the minimum protease concentration expected after manipulation) urinary proteins were completely degraded. Samples exceeding the threshold were analyzed by SDS-PAGE and Coomassie staining and showed either completely empty lanes or lanes with few bands, which enabled identification of the added proteases by mass spectrometry (either by in-gel digestion of the excised band or by determination of proteolysis and autolysis products of the enzymes ). By using the latter approach Kohler et al.  were able to report the presence of exogenous bacillolysin in two doping-control samples from elite athletes. Another approach was published by Lamon et al. (2007) , who suggested the use of protease-induced digestion of endogenous urinary albumin as a test for protease activity (revealed by Western blotting with an anti-albumin antibody after SDS-PAGE).
To protect the Epo contained in urine samples from enzymatic degradation, Tsivou et al. (2010) proposed addition of a stabilization mixture containing protease inhibitors (including, e.g., PMSF, pepstatin-A, trypsin inhibitor) and antimicrobial substances (sodium azide, pen-strep-fungizone) . As an alternative, Sanchis-Gomar et al. (2010)  considered cleaning the urethra by first collecting a small volume of urine and then collecting the urine for the A and B samples—a less expensive alternative for preventing athletes from using proteases.
Mass spectrometric methods
During the past two decades mass spectrometry (MS) of erythropoietin has been mostly focussed on the characterization of recombinant Epos. This has been largely because the amount of commercially available uhEpo has been very limited and its purity low. Consequently, most attempts, which had initially started with the idea of structurally characterizing uhEpo, ended with the characterization of rhEpos—a necessary basis for discovering possible differences from uhEpo. Hence, only a very limited amount of articles addressing these differences has so far appeared in print [102, 103, 104, 105].
using the tryptic T8 Epo peptide (54MEVGQQAVEVWQGLALLSEAVLR76) instead of the T6, T9, and T17 peptides used in the earlier publications; and
introducing a PEG-6000 precipitation step before immunoaffinity purification of the plasma.
The T8 peptide is common to rhEpo, NESP, and Mircera, and hence does not enable differentiation between these three Epo pharmaceuticals. The limits of detection and identification obtained were 0.1/0.05/0.3 ng mL−1 and 0.2/0.1/0.5 ng mL−1 for rhEpo/NESP/Mircera, respectively.
In 1996 Wrighton et al.  discovered, by phage display methodology, that small peptide molecules without sequence relation to Epo can also interact with the Epo receptor (Epo-R) causing the same receptor dimerization and signal transduction as observed for Epo. The peptides, called Epo mimetic peptides (EMP), were cyclic, because of an internal disulfide bond, and contained a fourteen amino acid minimum consensus sequence. One of these peptides (EMP1; 1GGTYSCHFGPLTWVCKPQGG20; the disulfide bridge is between C6 and C15, conserved amino acids are underlined) was further investigated. It was shown that covalent dimerization (either by a two-amino-acid linker via the C-termini  or by a bifunctional PEG-linker via the N-termini ) led to increased affinity for Epo-R. Hematide (Peginesatide, AF-37702; Affymax and Takeda Pharmaceutical) was developed on the basis of these discoveries and is currently in clinical phase 3. The chemical structure has been published by several authors [123, 124, 125]. According to these data Hematide is an EMP dimer containing three unusual amino acids (N-acetylglycine, 1-naphthylalanine, and N-methylglycine (sarcosine) at positions 1, 13, and 20, respectively) and two 20-kDa PEG groups. Doses used in clinical trials were in the range of ca 0.025 to 0.1 mg kg−1 every four weeks [126, 127]. Because of the large amount of PEGylation, doping with Hematide is probably best detected in serum or plasma. On the basis of the unusual amino acids and the rather high doses, mass spectrometric detection of Hematide for doping control purposes should be feasible. Mass spectrometric data for the monomeric peptide were published by Thevis et al. in 2009 .
For the immunological detection of Hematide refer to the section on ELISA-based methods.
Athlete biological passport
changes haematological data; and
that these changes are best monitored individually and longitudinally for each athlete.
Detection of EPO gene doping
Aside from attempting to directly detect the transgenic protein and possible differences by comparison with the non-transgenic protein, an alternative strategy is the direct detection of the transgenes, preferably not in the treated tissue but in blood. Beitler et al.  used the observation that transgenes usually lack the intron sequences normally present in human genes. A so-called single-copy primer-internal intron-spanning PCR method (spiPCR) was developed. The primers were designed in a way that their 3′ and 5′ ends only hybridize with two neighbouring exons, which are not separated by introns. Consequently, PCR amplification occurs only if the transgene is present. The method was developed to screen for six prime candidate genes (including Epo and vascular endothelial growth factors) in blood and was tested in a mouse model for vascular endothelial growth factor A (VEGF-A) . The transgene was delivered intramuscularly by an adeno-associated virus system. Only 20 μL blood was required and this enabled detection of the gene therapy for up to 56 days. A similar approach was reported by Baoutina et al. (2010) .
Apart from gene transfer, Epo gene expression also can be regulated by small orally administered molecules (e.g. transcription factor stabilizers). Prolylhydroxylase inhibitors (e.g. FG-2216, FG-4592; FibroGen; currently clinical phase 2 studies have been halted) stabilize hypoxia-inducible factor 1 (HIF-1α subunit) by preventing its enzymatic hydroxylation and subsequent degradation and thus increase Epo gene expression. Direct detection of the inhibitors might be possible by mass spectrometry .
Erythropoiesis-stimulating agents have been continually developed further since the first approval of recombinant Epo alfa for clinical use in 1989. This evolution fostered the development of new methods for detection of ESA doping and adaptation of existing methods to an ever changing situation. Recent advances have been made in the direct detection of Epo by gel electrophoretic methods, with the introduction of molecular mass-based methods (SDS-PAGE, SAR-PAGE) in screening and confirmation procedures. This strategy was especially useful for obtaining evidence of the abuse of Dynepo, NESP, and Mircera from urine and blood samples. Because of its increased sensitivity for Mircera, SAR-PAGE should preferably be used for serum and plasma samples instead of SDS-PAGE (one matrix one method approach). However, both methods are ideally suitable for confirming the presence of unstable (“active”) and “effort” urine samples as detected by IEF-PAGE. Targeted screening for the abuse of Mircera has been made possible with the development of an ELISA method for CERA. Another innovative method is the so-called “EPO WGA MAIIA” test, which is currently under evaluation for use as a screening method for the abuse of many different Epos. Adulteration of urine samples during collection by addition of proteases by the athlete has resulted in the development of procedures for protease detection and identification. The applicability of mass spectrometry for direct detection of NESP in human plasma and for rhEPO, NESP, and Mircera in horse plasma has been demonstrated in a series of publications between 2007 and 2010. However, these methods are not useful for obtaining evidence of the presence of rhEpo in human plasma. Because of the short serum half-life of rhEpos, indirect (marker) methods using blood data have been developed. The “Athlete Biological Passport” (haematological module) is the latest development in multivariable and longitudinal indirect testing. Its Bayesian statistical approach enables calculation of athlete-specific blood data limits and hence more efficient and targeted testing. Because of its open concept the passport is ready for inclusion of future advances in the detection of ESA doping by alternative methods, for example transcriptomics, proteomics, and metabolomics.
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