identification of zinc-alpha-2-glycoprotein binding to clone ae7a5 antihuman epo antibody by means...

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 2008; 43: 916–923Published online in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/jms.1444

Identification of zinc-alpha-2-glycoprotein bindingto clone AE7A5 antihuman EPO antibodyby means of nano-HPLC and high-resolutionhigh-mass accuracy ESI-MS/MS

Christian Reichel∗

Doping Control Laboratory, Austrian Research Centers GmbH-ARC, A-2444 Seibersdorf, Austria

Received 11 April 2008; Accepted 9 May 2008

The detection of doping with recombinant erythropoietins (Epo) by isoelectric focusing (IEF) and Westerndouble blotting strongly relies on the specificity of the detection antibody used. Currently a monoclonalmouse antibody (clone AE7A5) is used for that purpose. Despite its excellent sensitivity (amol range) theantibody shows some nonspecific binding behavior. However, the binding occurs outside the currentlyused pH range for evaluating erythropoietin IEF profiles. A shotgun proteomics approach is describedconsisting of preparative IEF on large-sized carrier ampholyte gels (pH 3–5), SDS-PAGE, Westernsingle and double blotting, on-membrane elution of intact proteins, on-membrane and in-solutiontryptic digestions, as well as nano-HPLC peptide separation and high-resolution high-mass accuracyESI-MS/MS peptide sequencing. The nonspecifically interacting protein could be identified as zinc-alpha-2-glycoprotein (ZAG). Confirmation analyses were performed using recombinant ZAG (rhZAG) and amonoclonal anti-ZAG antibody. It could be demonstrated that the binding of the monoclonal antihumanEPO antibody (clone AE7A5) to ZAG occurs in a highly concentration-dependant manner and that onlysamples containing increased amounts of urinary ZAG lead to a detectable interaction of the AE7A5antibody on Epo-IEF gels. Copyright 2008 John Wiley & Sons, Ltd.

KEYWORDS: erythropoietin; doping control; nonspecific binding; zinc-alpha-2-glycoprotein; clone AE7A5; proteomics

INTRODUCTION

Nonspecific binding of the monoclonal antihuman Epoantibody (clone AE7A5; R&D Systems, Inc.) used for thedetection of doping with recombinant erythropoietin by theworldwide-practiced isoelectric focusing (IEF) and Westerndouble-blotting method1 has been in discussion for severalyears. Publications in peer-reviewed journals addressedthis subject in 2005 and 2006 and led to some scientificargumentation on the specificity of the employed detectionantibody.2 – 6 However, under experienced users of the Epomethod several additional bands (usually 2–4) in the basicarea (nomenclature according to Ref. 7) of the pH 2–6 IEF-gel-caused scientific interest, because these bands couldonly be occasionally detected among the tested urinarysamples. Their relationship to Epo remained unclear foryears. Nevertheless, it was obvious that these bands didnot interfere with the endogenous urinary and recombinantEpo-IEF profiles and thus the evaluation of the profiles wasnot disturbed or questioned at all.2 Applying a shotgun

ŁCorrespondence to: Christian Reichel, Doping Control Laboratory,Austrian Research Centers GmbH-ARC, A-2444 Seibersdorf,Austria. E-mail: [email protected]

proteomics-based approach appeared to be the method ofchoice for solving this problem.

EXPERIMENTAL

MaterialsCarrier ampholytes (Servalytes 2–4, 3–4, 4–5, 4–6, and6–8) were from Serva (Heidelberg, Germany). Acry-lamide/bisacrylamide solution for IEF (PlusOne ReadySolIEF, 40% T, 3% C), urea, ammonium peroxidisulfate (APS),and N,N,N0,N0-tetramethylethylenediamine (TEMED), Tris,and glycine were from GE Healthcare (Uppsala, Sweden).For sodium dodecylsulfate polyacrylamide gel electrophore-sis (SDS-PAGE) separations BisTris-gels (NuPAGE, 10%T) together with lithium dodecyl sulfate (LDS) samplebuffer and 4-morpholinepropanesulfonic acid (MOPS) elec-trophoresis running buffer were used (Invitrogen, Carlsbad,CA). Devices for microfiltration (Steriflip) and ultrafiltra-tion (Centricon Plus-20, Centricon YM-30, Microcon YM-30),polyvinylidene difluoride (PVDF) membranes (Durapore,Immobilon-P) were from Millipore (Billerica, MA). Methanol(HPLC grade), acetonitrile (ACN) (HPLC hypergrade),glacial acetic acid, formic acid (FA), and water for liquid chro-matography mass spectrometry (LC-MS) (HPLC grade) werefrom Merck (Darmstadt, Germany). Reversed phase columns

Copyright 2008 John Wiley & Sons, Ltd.

Nonspecific binding of clone AE7A5 Epo antibody to ZAG 917

for peptide trapping (C18 PepMap100, 300 µm ð 5 mm,5 µm particle size, 100 A pore size) and nano-HPLC (C18PepMap100, 75 µm ð 15 cm, 3 µm particle size, 100 A poresize) were bought from Dionex (Sunnyvale, CA). Glass emit-ters for online nano-ESI (360 µm OD, 20 µm ID, 10 µm tipID) were from New Objective (Woburn, MA). Monoclonalmouse antibodies for the detection of erythropoietin (cloneAE7A5) and zinc-alpha-2-glycoprotein (ZAG; clone 35) wereacquired from R&D Systems (Minneapolis, MN) and BDBiosciences (San Jose, CA), respectively. The standard forhuman urinary erythropoietin (uhEpo; second internationalreference preparation) was from the National Institute forBiological Standards and Control (NIBSC, Hertfordshire,UK), standards for recombinant erythropoietin (rhEpo) fromthe European Directorate for the Quality of Medicines (BRP-Epo; Strasbourg, France) and Amgen (NESP; Thousand Oaks,CA). Recombinant zinc-alpha-2-glycoprotein (rhZAG) wasobtained from BioVendor (Modrice, Czech Republic). Alpha-1-antitrypsin, alpha-1-acid glycoprotein, trifluoroacetic acid(TFA), dithiothreitol (DTT), iodoacetamide (IAA), trifluo-roethanol (TFE), and ammonium bicarbonate were fromSigma-Aldrich (St. Louis, MO). Urine samples were eitherofficial samples taken from athletes for Epo-doping controlor samples received from healthy volunteers. Athletes andvolunteers gave their written consent to using the samplesfor research purposes. The project was approved by the localethics committee.

Electrophoretic separation and Western blottingIEF on carrier ampholyte (CA) slab gels was used forseparating endogenous urinary and recombinant humanerythropoietin isoforms (standards, urine samples). Twodifferent pH gradients, namely, pH 2–6 and pH 3–5,and two different gel sizes (24 ð 10 cm2, 24 ð 20 cm2) wereemployed. The method (gel casting, sample preparation,IEF) was essentially performed as described by Lasneet al.8. For large-sized gels (pH 3–5 XL and pH 2–6XL) electrophoresis conditions were adapted accordingly(Table 1). Interelectrode distances were 10 and 17 cm,respectively. The temperature of the cooling device was set to8 °C. After focusing, gels were blotted on PVDF membranesusing modified Towbin buffer (25 mM Tris, 192 mM glycine,no methanol) as described by Lasne.8,9 For chemiluminescentdetection of the specific and nonspecific binding of cloneAE7A5 anti-Epo antibody single and double blots wereused. Incubations with the anti-ZAG antibody (clone 35)were performed on first blots only and after the anti-Epoantibody was stripped by semidry blotting under acidicconditions (0.7% acetic acid, 0.8 mA/cm2, 10 min).

After incubation with a biotinylated secondary anti-body (ImmunoPure goat antimouse IgG; Pierce, Rockford,

IL) followed by several washing steps [0.5% low-fat milkin phosphate buffered saline, (PBS)] and a streptavidinhorseradish peroxidase (HRP) incubation step (ImmunoP-ure; Pierce, Rockford, IL), the membranes were washed withPBS. Chemiluminescence was achieved by incubation of theblots in a luminol-based substrate (West Pico; Pierce, Rock-ford, IL). All images were acquired with a CCD camera(epoCAM, ARC Seibersdorf research, Austria) and at vari-able exposure times depending on the initial signal intensity(e.g. 1, 2, 5, and 10 min).

SDS-PAGESDS-PAGE separations were performed on BisTris-gels (10%T, 1.5 mm) at constant voltage (200 V) and for 50 min(MOPS running buffer). The samples and standards wereheated at 70 °C for 10 min in LDS sample buffer and underreducing conditions. Between 20 and 35 µl of the denaturedprotein solutions were applied on the gels via GELoader tips(Eppendorf; Hamburg, Germany). After electrophoresis gelsand membranes (Durapore, Immobilon-P) were equilibratedin Towbin buffer (25 mM Tris, 192 mM glycine, 20% methanol)for 15 min and blotted at constant current (1 mA/cm2) for60 min (Trans-Blot SD Semidry Electrophoretic Transfer Cell;BioRad, Hercules, CA). All subsequent steps were doneexactly as described for the IEF gels.

On-membrane protein elutionBands showing nonspecific binding of clone AE7A5 anti-Epo antibody after IEF and double blotting were excisedfrom the first membrane after stripping the antibody anddrying the membrane. The PVDF-membrane pieces weretransferred to 2 ml Eppendorf tubes and incubated with40 µl one-fold LDS sample buffer at 95 °C for 5 min andunder reducing conditions (Thermomixer, 700 rpm). Aftercooling on ice and centrifugation at 16 100 rcf for 5 minthe eluates were directly applied on BisTris-gels (10% T,1.5 mm). Electrophoretic conditions were as described above.In order to show whether these bands resembled isoformswith approximately the same molecular mass, double blotsof the SDS-PAGE gels were performed. In addition, the firstmembranes were also incubated with the clone 35 anti-ZAGantibody.

On-membrane tryptic digestion of nonspecificallybound proteinsFor mass spectrometric identification of nonspecificallybound protein bands on the first membranes of both the IEFand SDS-PAGE separations were excised and transferredto 2 ml Eppendorf tubes. Subsequently 25 µl of a solutionof 0.2 µg/µl trypsin (sequencing grade modified; Promega,Madison, WI) in 25 mM Tris-HCl buffer pH 8.5 containing

Table 1. Focusing conditions for low- and high-resolution-IEF slab gels

Step pH 2–6 IEF gel (24 ð 10 cm2)pH 3–5 XL, pH 2–6 XL IEF gels(24 ð 17 cm2)

Prefocusing 250 V/150 mA/70 W/30 min 3500 V/40 mA/34 W/60 minFocusing 2000 V/131 mA/25 W/4000 Vh 3500 V/25 mA/25 W/1000 Vh

3500 V/25 mA/50 W/9000 Vh

Copyright 2008 John Wiley & Sons, Ltd. J. Mass Spectrom. 2008; 43: 916–923DOI: 10.1002/jms

918 C. Reichel

4 M urea were added and the tubes were incubated at 37 °Covernight (Thermomixer, 700 rpm). For mass spectrometricanalysis the tubes were sonicated for 2 min and 10 µl ofthe digest were mixed with 90 µl of 1% ACN containing0.05% TFA. The protocol was based on an in-solution trypticdigestion protocol10 and was adapted for on-membraneapplication as described above.

In-solution tryptic digestion of recombinantzinc-alpha-2-glycoproteinA stock solution (1 µg/µl) of rhZAG was prepared byreconstituting the lyophilized protein formulation with Milli-Q water. In order to remove interfering buffer substances,the protein was precipitated by adding cold acetone (�20 °C,150 µl) to 30 µl of the stock solution. After overnightincubation at �20 °C, centrifugation at 16 100 rcf for 5 min,and subsequent acetone removal, the pellet was dried for10 min at ambient temperature and resolubilized in 25 µlof 100 mM ammonium bicarbonate stock solution. Proteindenaturation and reduction of disulfide bridges was doneby adding TFE (25 µl) and DTT (1 µl, 200 mM) (60 °C, 60 min,Thermomixer, 400 rpm) according to a published protocol.11

The resulting free thiol groups were carbamidomethylatedby adding 4 µl of a freshly prepared solution of IAA (200 mM)and incubating in the dark at 25 °C for 60 min (Thermomixer,400 rpm). Excess IAA was removed by adding 1 µl ofthe 200 mM DTT solution (25 °C, 60 min, Thermomixer,400 rpm). Subsequently, 300 µl of Milli-Q water, 100 µlammonium bicarbonate stock solution (100 mM), and 0.6 µltrypsin solution (dissolved in 25 mM ammonium bicarbonatesolution at a concentration of 1 µg/µl) were added. Themixture was incubated at 37 °C (Thermomixer, 400 rpm)overnight (all digestion steps were followed the publishedprotocol11). For mass spectrometric analysis aliquots of thedigest were mixed 1 : 1 with 1% ACN containing 0.05% TFA.

LC-MS/MSTryptic digests were separated on a Dionex Ultimate 3000dual gradient nano-HPLC system (Sunnyvale, CA) in onlinepreconcentration mode. For peptide trapping a C18 PepMap100 column (300 µm ð 5 mm, 5 µm particle size, 100 A poresize) was used and was operated at a flow rate of 20 µl/min.After loading the peptides on the trap column salts wereremoved by washing the column with loading solvent (2%ACN with 0.05% TFA). After 3 min the trap column wasswitched in line with the analytical column (C18 PepMap100,75 µm ð 15 cm, 3 µm particle size, 100 A pore size), whichwas operated at a flow rate of 300 nl/min. Solvents A (2%ACN with 0.1% FA) and B (80% ACN with 0.1% FA) wereused as mobile phases. A linear gradient starting at 0% B andending at 80% B after 120 min was used. Then both columnswere washed with 100% B for 25 min and reequilibratedafter switching the trap column off line with 0% B andloading solvent, respectively, for a further 25 min. Injectionvolumes were 1 µl. The autosampler was operated at 4 °C.UV absorption was recorded at 214 nm (3 nl in line-flow cell).The column oven temperature was 25 °C.

Eluting peptides were sequenced with low-energy CIDand an LTQ-Orbitrap FT mass spectrometer (Thermo

Electron, Bremen). A Proxeon nano-ESI source (Odense,Denmark) equipped with a New Objective glass emitter(360 µm OD, 20 µm ID, 10 µm tip ID) for online nano-ESI (Woburn, MA) was used. The mass spectrometer wasoperated in positive-ion mode with a spray voltage of 2 kVand a capillary temperature of 160 °C. Data were acquiredin data-dependent mode (one full MS scan (m/z 300–2000)at a resolving power of R D 60 000 at m/z 400 followed byMS/MS scans of the three or five most intense ions in thelinear-ion trap) with a cycle time of approximately 1 s forthe top three experiment (one full MS scan experiment atR D 60 000 in the Orbitrap took ca 1 s and thus enabledthree parallel MS/MS experiments in the linear-ion trap).The lock-mass option of the orbitrap analyzer was used forall measurements and enabled online recalibration of theinstrument. The polydimethylcyclosiloxane background ionat m/z 445.120025 was used as lock mass. Mass accuracy wasusually better than 5 ppm. Dynamic exclusion was enabledwith 30 s exclusion duration. Automated gain control (AGC)target settings were 2 ð 105 for full MS scans in the FTanalyzer and 1 ð 104 for MS/MS scans in the linear-ion trap.The high-resolution instrument was chosen in order to enableonline charge-state determination of precursor ions and toadditionally use monoisotopic precursor mass informationfor subsequent bioinformatic analyses.12

BioinformaticsBioWorks 3.3 (Thermo Electron, San Jose, CA) was used forsearching the experimental tandem mass spectra (MS/MSdata) against the UniProt protein database (downloaded Jan-uary 2006). SEQUEST-search parameters were set as follows:trypsin (fully enzymatic, cleaving at both ends), two missedcleavage sites, 5 ppm peptide mass tolerance, monoisotopicprecursor mass, and 1 amu fragment-ions tolerance. Oxi-dized methionine was set as variable modification and incases where the protein mixture was treated with IAA beforetryptic cleavage carbamidomethylcysteine was set as fixedmodification. The top SEQUEST hit was manually confirmedaccording to the criteria proposed by Yates et al.13.

RESULTS AND DISCUSSION

Electrophoretic separation and Western blottingIEF of the same urine samples (pH 2–6, pH 2–6 XL, andpH 3–5 XL) showed that the nonspecific binding behaviorof clone AE7A5 anti-Epo antibody was neither influencedby the gel size nor the pH gradient used and thus wasreproducible (Fig. 1). Within these pH ranges usually threeto four extra bands were detected in the extreme basic areaof the gels after double blotting (Figs 1 and 2(a)).

However, it must be emphasized that not all samplesshowed these extra bands. Owing to the improved separationof the bands on the pH 3–5 XL gels these gels were used for allsubsequent SDS-PAGE and MS experiments. SDS-PAGE anddouble blotting was performed with urinary ultrafiltrationretentates which showed extra bands during IEF and resultedin electropherograms with one additional band. Its apparentmolecular mass was higher than the apparent mass of uhEpobut slightly lower than the mass of NESP (Fig. 2(d)).



Figure 1. Reproducibility of the nonspecific binding on pH 2–6 and pH 3–5 XL gels. Three different samples were run on the threedifferent gel types and on different days. Owing to the higher resolving power of the large-sized gels (pH 3–5 XL) compared to the pH2–6 gels preparative isolation of the nonspecifically detected bands was facilitated. Figure 1 shows the results for the small-sizedpH 2–6 and large-sized pH 3–5 XL gels. Bands numbered from 1 to 4 were the ones used for mass spectrometric identification.

Figure 2. Isolation of the unknown protein. (a) Isoelectric focusing on a pH 3–5 XL carrier ampholyte gel resulted in good separationof the four nonspecifically detected bands. (b) Excision of the bands from the first PVDF-membrane combined with on-membraneelution and (c) subsequent SDS-PAGE showed that all four bands had the same apparent molecular mass. (d) In comparison, directSDS-PAGE of the same urinary retentates led to a similar result but with the uhEpo bands still present.

On-membrane protein elutionElution of the three to four extra bands from one and thesame IEF-lane (Fig. 2(b)) followed by SDS-PAGE and doubleblotting showed identical apparent molecular masses forthese bands (Fig. 2(c)). Hence, it was most likely that thebands represented isoforms of one single protein.

LC-MS/MSA shotgun proteomics approach was used in order toidentify the protein causing the nonspecific binding of theclone AE7A5 antibody. After cutting the four bands out of

the first PVDF membrane the proteins were on-membranetryptically digested. Subsequent nano-LC-MS/MS runs andprocessing of the MS/MS spectra with Bioworks 3.3 softwareand the UniProt database revealed a series of possibleproteins. Among them ZAG was present in all four fractionsand showed highest sequence coverage (ca 52–62%) andidentification scores (SEQUEST protein score values betweenca 298–336; Table 2 represents the results of band numberone).

In order to confirm the identity of the protein,MS/MS spectra of a tryptic digest of rhZAG were also


920 C. Reichel

Table 2. UniProt search results for the MS/MS spectra of the four nonspecifically detected bands on the pH 3–5 XL IEF gel(algorithm: SEQUEST, Bioworks 3.3). Only the top 15 hits are listed. Bands 1–4 were cut out of the first PVDFmembrane and thendigested on-membrane with trypsin. Zinc-alpha-2-glycoprotein was the protein with the highest identification score and sequencecoverage. The P-value (P) is the probability of the best-matched peptide of the respective protein to be a false positive hit

Accessionnumber Protein Mr [kDa]

Sequencecoverage

[%] P Score

P25311 Zinc-alpha-2-glycoprotein precursor 33 851 53.2 1.32E-13 340.3P02760 AMBP protein precursor 38 974 19.9 2.22E-15 160.3O94919 Probable endonuclease KIAA0830

precursor54 981 30.6 3.53E-10 146.3

P01042 Kininogen precursor 71 900 12.7 1.71E-12 138.2P01009 Alpha-1-antitrypsin precursor 46 707 32.3 1.14E-10 124.3P02750 Leucine-rich alpha-2-glycoprotein

precursor38 154 30.0 1.25E-09 98.2

P11684 Uteroglobin precursor 9987 41.8 1.22E-14 90.3P02652 Apolipoprotein A-II precursor 11 168 49.0 2.79E-06 68.2P02765 Alpha-2-HS-glycoprotein precursor 39 300 11.7 3.75E-11 46.3P01834 Ig kappa chain C region 11 602 49.1 2.06E-12 40.3P02763 Alpha-1-acid glycoprotein 1 precursor 23 497 20.9 2.22E-14 20.3P41222 Prostaglandin-H2 D-isomerase

precursor21 015 8.4 5.08E-12 20.3

P02766 Transthyretin precursor 15 877 27.9 1.56E-07 20.3Q9H299 SH3 domain-binding glutamic

acid-rich-like protein 310 431 31.2 8.49E-06 20.1

P01857 Ig gamma-1 chain C region 36 083 10.0 1.49E-10 18.2

acquired and manually compared with the peptide spec-tra of the presumptive protein (Fig. 3). Detailed analy-sis of those peptides within the ZAG sequence whichwere not automatically identified by SEQUEST revealedthat they resembled peptides bearing N-glycosylation sites.Naturally these peptides were not part of the UniProtdatabase.

Binding of clone AE7A5 anti-Epo antibody tozinc-alpha-2-glycoproteinAfter successful identification of the unknown protein bymass spectrometry the binding behavior of clone AE7A5anti-Epo antibody was studied in greater detail. For thatpurpose both pH 2–6 XL and pH 3–5 XL IEF gels were used.Recombinant human ZAG (2 µg) was separated togetherwith uhEpo, BRP-Epo, and NESP (0.2 ng each). It could beclearly demonstrated that the anti-Epo antibody not onlybound to the various erythropoietins but also to severalrhZAG isoforms. Stripping and reprobing of the first blotwith the clone 35 anti-ZAG antibody (Fig. 4) showed that(1) AE7A5 only bound to the most abundant rhZAG isoformsand that (2) uhZAG was also present in uhEpo, but at alower concentration (the NIBSC-standard for uhEpo contains10 IU (80 ng) uhEpo and about 2 mg of other urinaryproteins).

The same experiment was repeated with samples ofathletes. Only urines containing an elevated amount ofuhZAG (roughly estimated by the relative chemilumi-nescence signal intensity obtained by the anti-ZAG anti-body reaction of the different samples) led to nonspe-cific binding of the clone AE7A5 antibody to uhZAG

isoforms (Fig. 5). Sequence homology searches were per-formed using the amino acid sequence of the peptide,which was used for generating the antibody (i.e. thefirst 26 amino acids of the N-terminus according tothe package insert of the antibody, which should beAPPRLICDSRVLERYLLEAKEAENIT), and the NCBI onlineprotein BLAST search tools (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). No sequence homologies with ZAG couldbe detected. Interestingly, the original publications of 1983and 1985 describing the development of the antibody14,15

mentioned a peptide sequence which was slightly differentfrom the sequence found in the UniProt database (above)for the first 26 amino acids of the processed precursorprotein – the C in position 7 of the published sequence(UniProt) was exchanged to an N, the N in position 24 to aK – thus resulting in APPRLINDSRVLERYLLEAKEAEKIT,the sequence used for generating the antibody way backthen). However, performing BLAST searches with thisaltered sequence also resulted in no homology to ZAG.This difference in the sequence was already mentioned inRef. 15. Today, it is common knowledge that N24 is usu-ally one of the three asparagine residues of Epo bearing anN-glycosylation.

Further experiments investigated the binding behavior ofthe antibody to two other highly abundant urinary proteins(alpha-1-antitrypsin, alpha-1-acid glycoprotein) on pH 2–6IEF and pH 3–5 gels. Theses proteins also showed highSEQUEST score values (Table 2). The tested protein amountwas 20 µg for the alpha-1-antitrypsin and ranged for thealpha-1-acid glycoprotein between 100 µg and 1 mg on gel.



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Figure 3. Manual confirmation of the SEQUEST-search result according to Ref. 10 and by comparative sequence analysis of theMS/MS spectra of human urinary ZAG and human recombinant ZAG. The example above shows the MS/MS spectra of precursormass 796.4 (3C) of uhZAG (above) and rhZAG (below). The sequence of the peptide was identified asNILDRQDPPSVVVTSHQAPGEK.

No nonspecific binding could be detected for both proteins.However, it must be stressed that the tested proteins were theproteins isolated from human plasma and consequently werenot completely identical to the ones found in human urine.A more detailed study on these and further highly rankedproteins is currently being performed. An investigation of apossible correlation between the uhZAG concentration andthat of Epo was not part of this study.

CONCLUSION

It could be demonstrated that ZAG – which is an abundantprotein in urine and which is present in many body fluidsand tissues – was present in all of the investigated urinary

samples screened for Epo-doping. It was also detectable inthe reference standard used for human endogenous urinaryerythropoietin (NIBSC). Relatively little is known about thebiological role of ZAG. Among the functions described inliterature are its involvement in lipid metabolism (binding offatty acids, lipid catabolism16), as well as regulatory rolesin cell adhesion and cell proliferation.17 The expressionrate of ZAG is also altered in certain types of cancer (e.g.breast carcinomas18), and kidney diseases.19 From its domainstructure it is related to immunoglobulins and MHC Class Iproteins.20

The binding of the monoclonal antihuman EPO antibody(clone AE7A5) to ZAG occured in a highly concentration-dependant manner. Consequently, only samples rich in


922 C. Reichel

Figure 4. Separation of BRP-Epo and NESP, uhEpo, rhZAG, and a mixture of uhEpo and rhZAG (lanes 1, 2, 4, 5 from left to right) ona pH 2–6 XL IEF gel (double blots). The first PVDF membrane was incubated with clone AE7A5 anti-Epo antibody, stripped and thenreincubated with clone 35 anti-ZAG antibody. The most intense uhZAG isoforms were observed in the region where the nonspecificbinding usually occurred for the urinary samples.

Figure 5. Samples of athletes were screened for Epo on a pH3–5 XL gel. (a) Nonspecific binding of the anti-Epo antibody(clone AE7A5) occured on few samples. (b) Stripping andreprobing of the first membrane with the anti-ZAG antibody(clone 35) revealed that uhZAG was present in all samples butAE7A5 bound only to those urines which had elevatedamounts of uhZAG.

urinary ZAG caused detectable binding of the AE7A5antibody to ZAG and thus led to the unexplained extrabands on the Epo-IEF gels.

REFERENCES

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3. Franke WW, Heid H. Pitfalls, errors and risks of false-positiveresults in urinary EPO drug tests. Clinica Chimica Acta 2006; 373:189.

4. Beullens M, Delanghe JR, Bollen M. False-positive detection ofrecombinant human erythropoietin in urine following strenuousphysical exercise. Blood 2006; 107: 4711.

5. Lasne F. No doubt about the validity of the urine test fordetection of recombinant human erythropoietin. Blood 2006; 108:1778.

6. Catlin D, Green G, Sekera M, Scott P, Starcevic B. False-positiveEPO test concerns unfounded. Blood 2006; 108: 1778.

7. Catlin D, Nissen-Lie G, Howe C, Pascual JA, Lasne F, Saugy M.WADA Technical Document TD2007EPO, http://www.wada-ama.org/en/ [Accessed 2008].

8. Lasne F, Martin L, Crepin N, de Ceaurriz J. Detection ofisoelectric profiles of erythropoietin in urine: differentiationof natural and administered recombinant hormones. AnalyticalBiochemistry 2002; 311: 119.

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identification of zinc-alpha-2-glycoprotein binding to clone ae7a5 antihuman epo antibody by means...

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