Tissue transglutaminase ELISA positivity in autoimmune diseaseindependent of gluten-sensitive disease
Miklós Sárdy a,⁎, Márta Csikós a, Christof Geisen b, Klaudia Preisz a, Zoltán Kornseé a,Erika Tomsits c, Ulrich Töx d, Nicolas Hunzelmann e, Jörgen Wieslander f,
Sarolta Kárpáti a, Mats Paulsson g, Neil Smyth g
a Department of Dermato-Venereology and -Oncology, Semmelweis University, H-1085 Budapest, Mária u. 41., Hungaryb Institute for Clinical Chemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Street 9, D-50924 Cologne, Germany
c Department of Pediatrics II, Semmelweis University, H-1094 Budapest, Tûzoltó u. 7-9, Hungaryd Department of Internal Medicine IV, Medical Faculty, University of Cologne, Joseph-Stelzmann-Street 9, D-50924 Cologne, Germany
e Department of Dermatology, Medical Faculty, University of Cologne, Joseph-Stelzmann- Street 9, D-50924 Cologne, Germanyf Wieslab Co., Ideon, S-223 70 Lund, Sweden
g Center for Biochemistry and Center for Molecular Medicine, Medical Faculty, University of Cologne, Joseph-Stelzmann-Street 52, D-50931 Cologne, Germany
Received 18 April 2006; received in revised form 1 August 2006; accepted 2 August 2006Available online 14 August 2006
Celiac disease (CD) is a common small bowel disorderassociated with a persistent intolerance to gluten and concom-itant immune and autoimmune (AI) phenomena. Changes in CDare not confined to the small intestine and it may be considered asystemic AI disease with frequent intestinal and infrequent (orrarely diagnosed) extraintestinal manifestations. In a few indi-viduals, CD is associated with dermatitis herpetiformis (DH), anAI skin disease characterized by polymorphic eruptions withunderlying granular IgA deposits occurring in the papillary
dermis. As CD and DH share very similar jejunal pathology,identical genetic background, similar pathomechanism, diag-nostic analysis, and dietary possibilities for therapy, the term‘gluten-sensitive disease’ (GSD) will be applied to both theseconditions.
The diagnosis of GSD is based on the characteristic histolog-ical and immunohistochemical changes seen in jejunal and/orskin biopsies [1]. However, serological tests may also be helpfulas they offer a less invasive and cheaper alternative. With theidentification of tissue (cellular) transglutaminase (TGc, alsocalled TG2) as the endomysial autoantigen of CD [2] and DH [3],a diagnostic ELISAwas produced giving a high sensitivity and aspecificity of above 90% after optimization using calcium-activation of the enzyme [4–7] yielding as high a performance asthe serological ‘gold standard’, the endomysium Ab (EMA) test.
In our and other investigators' studies, certain groups of AIpatients without GSD showed significant reaction in the TGcELISA. This suggested either a more general role for TGc in AIprocesses or a technical problem. The aim of this study was toconfirm which if either of these 2 possibilities was the cause,and investigate the reason for this unexpected positivity. UsingTGc ELISAs, sera from patients with a broad spectrum of AIdiseases were compared with those from patients with GSD aswell as non-AI diseases involving organs with enhanced apo-ptosis and cell lysis and/or putative secondary AI processes, e.g.hepatitis or malignancies, where release of TGc could theo-retically lead to anti-TGc Ab formation. Controls of healthyvolunteers and patients with unrelated diseases were used.
2. Patients and methods
2.1. Sera and patients
The sera were from the Gastroenterological Departments ofInternal Medicine or Pediatrics and the Department ofDermato-Venereology of the Semmelweis University, theDepartments of Internal Medicine I–IVof the Medical Facultyof the University of Cologne, and from the Laboratory forAutoimmune Diseases of the Wieslab Company, Sweden.Serum samples were taken from 304 AI patients, and 95patients with GSD including untreated CD and DH patients(for numbers and diagnoses see Table 1). Sera from patientswith psoriatic arthritis, hepatitis C, and different malignancieswere also studied (Table 1). The term ‘malignancies’ includes2 patients with primary liver carcinoma, 3 patients with thyroid
gland carcinoma, and single cases with neuroblastoma, gastriccarcinoma, hypernephroma, rectal adenocarcinoma, plasmo-cytoma, and a tumor of unknown origin giving metastases inliver, mediastinal lymphnodes, and bones. Twenty-six of the84 control patients were healthy individuals while 58 hadvarious non-AI diseases (Table 2). In total, 605 serum sampleswere analyzed. The mean age and sex of the patient groups arepresented in Table 1. All serum samples were stored at −78 °Cuntil assayed.
The diagnoses of the AI diseases were confirmed by thefollowing guidelines: CD: jejunal biopsy pathology and EMA-positivity [1]. DH, pemphigus vulgaris and bullous pemphigoid:conventional skin histology, direct and indirect immunofluores-cence. Ulcerative colitis and Crohn's disease: a combination ofclinical, radiological, endoscopical, and histological signs.Goodpasture's syndrome: clinical symptoms together with im-munofluorescence showing autoantibodies against glomerularbasement membrane. Wegener's granulomatosis: 1990 Ameri-can College of Rheumatology criteria; in addition, positivity forPR3-ANCA Abs [8]. Rheumatoid arthritis, SLE and systemicsclerosis: American College of Rheumatology (former Ameri-can Rheumatism Association) criteria [9–11]. Primary
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antiphospholipid syndrome: clinical symptoms; at least one ofthe typical laboratory changes (positivity for lupus anticoagulantor anti-cardiolipin Abs of IgG or IgM class) detected twice in aperiod of at least 12 weeks; the absence of any signs or symp-toms of other AI disorders, infections, malignancies or adverseside effects of medicaments. Psoriatic arthritis: Wright andMoll's criteria [12]. Hepatitis C: positive PCR analysis and/orpositive serology together with clinical symptoms of liverdisease including increased transaminase levels and/or positiveliver histology. Malignant tumors: histology.
2.2. Total serum IgA measurement, gliadin ELISA and EMAtest
The EMA test was performed with each serum from patientswith CD or DH. In addition, the total serum IgA level wasmeasured, and anti-gliadin Ab (AGA) and EMA tests carriedout on a randomized subgroup of AI patients having IgA Absagainst human TGc. Those chosen suffered from the followingdisorders (number of patients in parentheses): SLE (17),rheumatoid arthritis (9), hepatitis C (15), psoriatic arthritis (3),progressive systemic sclerosis (19), primary antiphospholipidsyndrome (4) and malignancy (3).
The total serum IgAwas measured using the Tina-quant IgAkit (Roche Diagnostics GmbH, Mannheim, Germany), the IgAAGA were detected with the Bindazyme Anti-gliadin IgA kit(Binding Site Ltd, Birmingham, England) according to theinstructions of the manufacturers. The total IgAwas consideredincreased when N4.9 g/l in men and N4.5 g/l in women, thecutoff of AGA ELISA was set according to the recommenda-tions of the manufacturer. Serum IgA EMAs were measured onmonkey esophagus sections by an indirect immunofluorescencemethod described previously [7].
Sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE), immunoblotting, and mass spectometry wasperformed as already described [7].
2.3. TGc
Human TGc was expressed recombinantly either in bacteria(and obtained as a kind gift from Dr. Róbert Király and Prof.László Fésüs, University of Debrecen, Hungary), or in humancells as described before [7].
2.4. Immunoblot testing AI sera for reactivity
Purified TGc or cell lysates of 293-EBNA human embryonickidney cells expressing human TGc were run on an SDS-PAGEgel and transferred to nitrocellulose membranes. After block-ing, the membranes were incubated with sera diluted 1:25 in50 mmol/l Tris, 150 mmol/l NaCl, pH 7.4 containing 1 g/l non-fat milk powder and 0.5 ml/l Tween 20 for 1.5 h at roomtemperature. For detection of bound IgA antibodies, membraneswere incubated with horseradish peroxidase-labeled rabbitAbs directed against human IgA (Dako), diluted 1:1000 in50 mmol/l Tris, 150 mmol/l NaCl, pH 7.4 containing 1 g/l non-fat milk powder and 0.5 ml/l Tween 20 for 1 h at room tem-
perature. Bound secondary antibodies were detected using theenhanced chemiluminescence system (ECL Kit, Amersham).
2.5. Purification of human TGc with anion exchangechromatography
After purification by streptavidin affinity chromatography,TGc was further purified by anion exchange chromatography.The sample containing affinity purified human TGc was diluted1:1 with cold (4 °C) water, and cleared of particulate materialby ultracentrifugation at 210,000 g for 20 min at 4 °C. Then thesupernatant was passed over a Mono Q HR 5/5 column(Amersham Pharmacia Biotech) attached to an FPLC system,and equilibrated with sterile filtered 20 mmol/l Tris/HCl,pH 7.5. After extensive washing with equilibration buffer, TGcwas eluted by 20 mmol/l Tris/HCl, pH 7.5 containing a gradientof 0–1 mol/l NaCl. Fractions (1 ml) were collected, and thepurification controlled by Coomassie- and silver-stained SDS-PAGE.
2.6. TGc ELISA
The recombinant human TGc ELISA method was performedas described previously [7]. The different TGc preparationswere used in the same way (1 μg of protein per well). All serumsamples were examined in duplicate or triplicate, and a negativeand 2 positive sera were included in each assay. The Ab con-centrations were expressed in arbitrary units (AU), i.e., as per-centages of the positive reference serum. In this study, we usedthe same positive reference serum as previously [7].
The anti-TGc sandwich ELISAwas done as follows: 96 wellmicrotiter plates (NuncMaxiSorp)were coatedwith 2.5μg F(ab′)2fragment of sheep anti-mouse IgG Abs (Sigma C-2181, adsorbedwith human serum proteins) in 100 μl of 50 mmol/l Tris/HCl,pH 7.5 per well at 4 °C overnight (12–14 h), followed byincubation of 300 ng mouse monoclonal Abs against TGc(Neomarkers, Ab-3 [a mix of Abs CUB7402 and TG100]) in100 μl of 50 mmol/l Tris/HCl, pH 7.5 per well for 1 h at roomtemperature. The wells were blocked by 1 g/l bovine serumalbumin in 100 μl of 50 mmol/l Tris/HCl, pH 7.5 for 2 h at roomtemperature. Up to this step, the wells were washed with50 mmol/l Tris/HCl, pH 7.5, whereas in each later step, the wellswere washed with 50 mmol/l Tris/HCl, pH 7.5 containing10 mmol/l EDTA, 1 ml/l Tween 20, and 140 mmol/l NaCl(TETS). Human TGc was bound to the wells by incubating 1 μg/well diluted in 100 μl of TETS containing 5 mmol/l CaCl2 for1.5 h at room temperature. Sera were diluted 1:250 with TETS,and incubated on the plates for 1.5 h at room temperature. BoundIgA was detected by horseradish peroxidase-conjugated Abagainst human IgA (Dako), diluted 1:4000 in TETS, andincubated for 1 h at room temperature. The color was developedby 100 μl of 60 mg/l 3,3′,5,5′-tetramethylbenzidine substrate in100 mmol/l sodium acetate, pH 5.6, containing 0.15 ml/l H2O2
for 5 min at room temperature. The reaction was stopped byadding 100 μl of 200 ml/l H2SO4. The absorbance was read in anELISA reader at 450 nm.
Fig. 2. Correlation of the total IgA level with the IgA Ab concentration againstgliadin. On the x-axis are total IgA levels given in g/l, on the y-axis are units/mlmeasured by the anti-gliadin Ab ELISA.
Fig. 1. Box and whisker diagram showing the serum IgA Ab concentrationsagainst the human TGc. The lower and upper edges of the boxes represent the25% and 75% percentiles, respectively. The median is indicated by a horizontalline through the box. The lower and upper whiskers represent the 5% and 95%percentiles, respectively. The arbitrary cutoff level for positivity is drawn bydashed line at the AU of 18. Abbreviations see Table 1.
The results of the TGc ELISAs did not show Gaussiandistribution, thus for a statistical description, the results fromthe different patient groups are presented as medians with95% confidence intervals (95% CI) [13], and for comparison,Mann–Whitney's non-parametric, unpaired, 2-tailed test wasused [14]. For describing correlations, Spearman's correlationcoefficient with its 95% CI and correlation analysis for data ofnon-normal distribution were used calculating 2-tailed p values[13,14].
3. Results
3.1. TGc ELISA
The median serum Ab concentrations against the human TGcare shown in Fig. 1. Those for the patients with CD and DHwere 89.5 AU (n=39, 95% CI: 74.2–98.3) and 63.5 AU (n=56,95% CI: 51.3–76.1), respectively. The difference between the 2forms of GSD was significant (p=0.0004), however the 95%CIs overlapped. One patient with CD (EMA-positive) and 2with DH (both EMA-negative) fell below the cutoff of 18 AU inthe TGc ELISA. No healthy individuals had TGc Ab levelsgreater than the cutoff, however, five control patients had Abconcentrations just greater, in the range of 18.5–22.1 AU. Thesehad been diagnosed as having (number of patients in paren-theses): ichthyosis (3), retarded growth of non-gastrointestinal
Table 3Frequency of increased total IgA level as well as AGA and EMA positivity of AIsera positive for human TGc ELISA
origin (1), primary adult lactose intolerance (1). Comparing CDand DH patients with the controls, the sensitivity and specificityof the human TGc ELISA was 96.8% and 94.0%, respectively.
Overall 49% of all non-GSD AI sera were positive in thehuman TGc ELISA. In some individual diagnostic groups therewere low median levels with increased titers only in a fewpatients (e.g. bullous pemphigoid and pemphigus vulgaris),while other groups had high median Ab levels (e.g. Wegener'sgranulomatosis and Goodpasture's syndrome) (Fig. 1). Thedifference between the median titers of the AI and the controlsera was significant in every case with the exception of thepemphigus vulgaris sera (bullous pemphigoid, p=0.012; pem-phigus vulgaris, p=0.152; SLE, p=0.0005; antiphospholipidsyndrome, p=0.0002; for each of the other groups pb0.0001).
Significant differences were also found in serum titers be-tween controls and patients with hepatitis C (pb0.0001), pso-riatic arthritis (p=0.0065), and malignancies (p=0.0005).Altogether 25% of the sera from patients with hepatitis C,psoriatic arthritis, and malignancies were positive in the humanTGc ELISA. While in most of the disease groups, the 95% CIs
Fig. 3. Correlation of the total IgA level with the IgA Ab concentration againstthe human TGc. On the x-axis are total IgA levels given in g/l, on the y-axis arearbitrary units measured by the human TGc ELISA. Note the relatively highscatter of the values.
Fig. 4. TGc preparation purified with one step streptavidin affinity chromatog-raphy showing significant amounts of impurities. I, SDS-PAGE visualized withCoomassie-staining; II, the same gel overdeveloped with silver stain. Thesample loaded onto the gel contained ∼3 μg of human TGc.
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confirmed the difference to the controls as being significant, the95% CIs overlapped in three (bullous pemphigoid, psoriaticarthritis, and malignancies).
Two possibilities could explain the high number of positiveresults: either the unexpected presence of circulating anti-TGcAbs in the serum samples from a wide range of different dis-eases, or false positivity.
3.2. Total serum IgA, AGA ELISA and EMA test
To examine the origin of reactivity, 70 of the non-GSDpatients with TGc serum Ab levels above the cutoff were chosenat random for the measurement of total serum IgA levels,and the detection of AGA and EMA. Fifteen (21.4%) had in-creased total IgA levels, although one of these was borderline(Table 3). While increased total IgA levels occurred in mostdiagnostic groups, it is of note that it was not seen in any patientwith primary antiphospholipid syndrome, while it was mostcommon in patients with rheumatoid arthritis (4/9) andmalignancies (2/3) (Table 3). No patient had IgA-deficiency.
IgA AGA-positivity was found in eight of the 70 patients(11.4%), one or two AGA-positive sera being found in eachdiagnostic group, again with the exception of patients with
Fig. 5. Immunoblot of 26 sera having a positivity of N25 AU in the TGc ELISA. TheTGc in significant amounts. Positions of molecular mass markers (kDa) are indicatdifferent gel runs, and although certain lanes were digitally modified (slight vertical sin the level of bands corresponding to the same protein are possible. Diagnoses: A–C,pemphigus vulgaris; O–P, bullous pemphigoid; Q–S, hepatitis C; T–U, systemic scleGoodpasture syndrome; Y, psoriatic arthritis; Z, Crohn disease. The recombinant hu(corresponding to a protein of ∼85 kDa, see arrow, Fig. 4 and [7]).
primary antiphospholipid syndrome. Five of these eight seraalso had an increased total IgA. The correlation of total IgAlevels with the AGA concentrations was significant, howeverthere was a large degree of scatter (pb0.0001; rS=0.6451; 95%CI= 0.4832–0.7641; Fig. 2).
None of the sera was positive for EMA. However, some serafrom patients with SLE and hepatitis C showed signals on themonkey esophagus sections arising from nuclei, epithelial peri-cellular and/or reticular structures different from the endomy-sium. Twelve of the 15 sera (80%) with increased total IgA hadmarkedly increased IgA Ab concentrations against the humanTGc (above 25 AU), and 58.8% of the sera giving over 30 AUin the TGc ELISA had increased total IgA. The human TGcELISA results correlated significantly with the total IgA level(p=0.0026), but the scatter of the data was high (rS=0.3544;95% CI=0.1303–0.5441) (Fig. 3). The same was true for thecorrelation of the TGc ELISA results with the AGA concentra-tions (p=0.0034; rS=0.3454; 95% CI=0.1201–0.5367).
In conclusion, the data suggested that the TGc ELISA posi-tivity was unrelated to the presence of EMA antibodies butcorrelated with the total IgA levels and so presumably re-presented a false positivity.
3.3. Experiments aimed at finding the cause of false-positivesignal
To find out, why the AI sera gave false positivity, 26 AI serawith IgA antibody concentrations greater than 24 AU in thehuman TGc ELISA were chosen at random. The experimentsdescribed below were not performed with the same 26 AI sera,but each type of experiment (e.g., all ELISAs) was carried outwith the same set.
3.4. Immunoblot for testing reactivity of sera
To test the possibility of reactivity with minor protein con-taminants present in the purified human TGc preparation, 26 AIsera were tested for IgA immunoreactivity against both theunpurified cell lysate of 293-EBNA embryonic kidney cells
antigen is the lysate of human embryonic kidney cells (293-EBNA) expressinged on the left, but they show approximate values as the lanes are the result oftretching or shrinking without addition or deletion of any band), small variationsCD; D–E, DH; F–G, malignancies; H–J, SLE; K–L, rheumatoid arthritis; M–N,rosis; V, Wegener's granulomatosis; W, primary antiphospholipid syndrome; X,man TGc has a molecular mass of 78.4 kDa, but it runs slower than expected
expressing TGc and the purified TGc preparation. The purifiedTGc preparation gave a single band when analysed on aCoomassie-stained gel, but showed contaminating proteinsupon overdeveloped silver staining, see Fig. 4. Most of the seraclearly reacted with several bands in the cell lysate demonstrat-ing autoreactivity against a number of different proteins (Fig. 5).Some of the sera did not show a band corresponding to themolecular mass of TGc. Moreover, when testing the purifiedTGc, only 2 CD sera showed strong positivity corresponding tothe size of TGc. The other sera reacted with bands other thanTGc, or they did not react with any protein in the purifiedsample (data not shown).
3.5. Further purification of TGc
To further test the role of protein contaminants, we set up anELISA using a coat of TGc purified by additional anion ex-change chromatography. Even after this second chromatogra-phy step, none of the eluate fractions contained perfectly pureTGc when visualised on a silver-stained SDS-PAGE gel (datanot shown). However, 7 of 26 (27%) AI sera showing falsepositivity in the human TGc ELISA reacted with fractions notcontaining TGc (data not shown), the major component of these
Fig. 6. Reactivity of sera in the different ELISAs. Connected dots represent individ140 mmol/l NaCl in the buffer for serum incubation; III, anti-TGc sandwich ELISA; IVpresented from the following diagnostic groups: A, GSD; B, GSD on a gluten-free dSee explanation for the numbered sera in the text.
fractions being determined by mass spectrometry tryptic peptidefinger printing to be heat shock protein 70. In conclusion, theresults above suggested that the false positivity was due toreactivity with impurities in the TGc preparations.
3.6. TGc ELISA with higher NaCl concentration
In order to decrease non-specific binding of IgA to the ELISAwells, studies with 26 highly positive AI sera were performedusing additional NaCl to reach a concentration of 140 mmol/l inthe buffer used for dilution of the sera and incubation in theELISA wells (TETS). Controls (for this and all further ELISAexperiments) were chosen so that true- and false-positive as wellas true- and false-negative sera were deliberately included giving(maintaining the cutoff at 18 AU) a baseline sensitivity andspecificity of 92.9% (95%CI, 66.1–99.8%) and 32.5% (95%CI,18.6–49.1%), respectively. To find fine differences, GSD serawith borderline reactivity were chosen in greater number andGSD patients on a gluten-free diet were also included, howeverthe latter were excluded from calculations of sensitivity andspecificity.
In the ELISA using 140 mmol/l NaCl, the titers of AI serabecame significantly lower (Pb0.0001) whereas those of GSD
ual sera tested in the following assays: I, original ELISA [7]; II, ELISA with, ELISA coated with TGc expressed in bacteria. For better visualization, sera areiet; C, controls having unrelated disease or healthy individuals; D, AI disorders.
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sera did not change significantly, although the titers of GSD serahaving low reactivity against TGc (18–25 AU) in the originalELISA decreased, remaining just above the (reduced) cutoff(Fig. 6A–D and Fig. 7 I–II.). However, the titer reduction of AIsera did not change diagnostic performance considerably (seeROC curve and AUC in Fig. 7 II.): keeping the sensitivity of92.9%with a cutoff of 16 AU, specificity only improved slightly(35.0%; 95%CI, 20.6–51.7%). Given these results, non-specificIgA adsorption was one but not the only reason for the false-positive results.
3.7. Anti-TGc sandwich ELISA
To ensure reactivity of sera exclusively with TGc, an anti-TGc sandwich ELISA was performed coating the ELISA wellswith anti-mouse immunoglobulin Abs followed by incubationwith mouse monoclonal Abs against TGc, and then blockedas detailed in the methods (in a separate assay, reactivity withthe blocking protein BSA was examined). Non-specific ad-hesion of contaminant proteins was prevented by the use of140 mmol/l NaCl and 1 ml/l Tween 20. In this assay, mostAI sera became negative whereas most GSD sera remainedpositive (Fig. 6A–D) improving diagnostic performance(Fig. 7I. and III.). However, only a sensitivity of 78.6%(95% CI, 49.2–95.3%) and a specificity of 85% (95% CI,70.2–94.3%) could be achieved, because the signals of threeGSD sera with low reactivity to TGc were reduced below thecutoff, and three of the negative controls (Fig. 6C, sera Nos. 6–8)
Fig. 7. ROC curves of different ELISAs (only sera shown in Fig. 6A, C, and D werethe buffer for serum incubation; III, anti-TGc sandwich ELISA; IV, ELISA coated w
as well as 2 AI sera (Fig. 6D sera No. 9–10) showed highpositivity. Substantial increase of signal was also observed incertain GSD sera (Fig. 6A and B, sera No. 1–4). Sera Nos. 1 and 4were negative in the original ELISA, and became highly positive.No. 1 was the serum of a young girl having EMA- and TGc-negative, but biopsy-proven CD. No. 4 was a formerly EMA- andTGc-positive patient being on a gluten-free diet at the time ofcurrent test. Sera Nos. 2 and 3 belonged to 2 DH patients bothbeing negative for EMA, but weakly positive for human TGcELISA. No. 5 was the EMA-negative, but human TGc-positiveserum of a first degree relative to a CD patient participating on ascreening program but lost for follow-up. The clinical diagnosesof Nos. 6, 7, and 8 (which were negative in all tests but the anti-TGc Ab ELISA) were primary adult lactose intolerance, growthretardation (and heterozygotic twin of a CD patient), and cowmilk sensitivity, respectively. Unfortunately, we were unable toinvite these individuals for a retesting. Nos. 9 and 10 were sera ofpatients having Goodpasture syndrome and systemic sclerosis,respectively, but the latter serum was slightly positive in anindependent ELISA coated with BSA, hence its high titer may beexplained in part by reactivity with BSA. The patient from whomserum No. 11 originated had SLE. These latter three sera werenegative for EMA.
3.8. ELISA using TGc expressed in bacteria for coating
To further test the possible role of impurities, ELISA plateswere also coated by human TGc produced recombinantly in
used for calculations). I, original ELISA [7]; II, ELISAwith 140 mmol/l NaCl inith TGc expressed in bacteria.
Table 4AI diseases having been suggested to be associated with GSD (see Refs. [16–23]and Refs. therein)
bacterial cells which allowed to examine the effect of non-human protein contaminants to reactivity of AI sera. No block-ing was used, and again, non-specific adhesion to contaminantproteins was prevented by 140 mmol/l NaCl and 1 ml/l Tween20. Also in this assay, most AI sera became negative whereasmost GSD sera remained positive (Fig. 6A–D) improvingdiagnostic performance (Fig. 7I. and IV.). However, this highspecificity of 94.9% (95% CI, 82.7–99.4%) was accompaniedby a decreased sensitivity of 64.3% (95% CI, 35.1–87.2%),because low titer GSD sera (titer b30 AU) showed diminishedreactivity. Thus it was not evident that the reduction of reactivityof AI sera was due to the absence of human protein contami-nants or overall loss in sensitivity.
4. Discussion
Previous work has shown contradicting results as to thecorrelation of TGc Abs with other diseases, a number of authorsreported false-positive results when using the TGc ELISA. Thebroad spectrum of diseases linked with TGc ELISA positivitysuggested either a general association of TGc with autoimmu-nity and cell destruction, or widespread technical problems.Hence we decided to test the TGc ELISA system developed byour group [7] using sera from a large number of patients with abroad spectrum of diseases and theoretically likely to developanti-TGc Abs. It should be noted however that this patientpopulation did not reflect the clinical situation in which TGcELISA is usually requested. We found that almost half of thepatients with non-GSD AI disease had appreciable titers in theTGc ELISA, while healthy controls were negative. (IgG Abswere not investigated in this study but may be significant andshould be analyzed in the future.) Further purification did notseparate TGc perfectly from small amounts of contaminants;however, it was an instructive experiment supporting that in-deed some AI sera reacted with proteins other than TGc.
The ratio of false-positive sera was very high if compared toprevious studies (see details below). The main reason for this is,besides using highly reactive AI sera, that the human TGcELISA cutoff was set at 18 AU according to previous ROCanalysis based on a different set of control patients [7], whichwas possibly inadequate given that similarly high false-positiverates were also observed in other ELISA systems in which thecutoff was set too low [15]. However, when we increased thecutoff to 25 AU raising the specificity to 100% with respect tothe control group, 25% of all AI sera still remained false-posi-tive. While certain TGc ELISA positive AI patients possiblyhad GSD (it being a common disease with a prevalence between1:100 and 300 in Europe), and especially as GSD associateswith various AI disorders (Table 4) [16–23], such high numbersare extremely unlikely in the absence of EMA immunoreactiv-ity. For this reason and on ethical grounds, the definitive iden-tification of GSD, small intestinal biopsy, was not performed inAI patients.
TGc ELISA positivity is common in non-GSD patients, inparticular in studies using the relatively impure guinea pig TGcas antigen. In one previous report [4], 5 of 6 non-GSD (andEMA-negative) patients with false-positive sera suffered from
AI diseases (SLE, primary biliary cirrhosis, and Crohn's dis-ease). In another, false-positives were found in 15% of inflam-matory bowel disease (ulcerative colitis and Crohn's disease), in36% of chronic liver disease (alcohol cirrhosis, primary biliarycirrhosis, primary sclerosing cholangitis, and AI hepatitis) andin 21% of type I diabetes patients [24]. The human TGc had amuch higher specificity. For instance, Carroccio et al. found ahigh false positivity (16%) in patients with chronic viral hepa-titis when using guinea pig, but not human TGc [25]. Clementeet al. also found false positivity in 50% of AI hepatitis orprimary biliary cirrhosis patients' sera when using guinea pigrather than human TGc [26]. However, the human antigen didnot result in a perfect specificity either, as method-dependentfalse positivity in different human TGc ELISAs was a strikingfinding also in cirrhosis entities of non-AI origin [27], and falseIgA and/or IgG anti-human TGc ELISA positivity was shown in1.9% of 618 AI patients' sera with only 0.3% of patients beingpositive for EMA and jejunal biopsy [28]. The range of dis-orders associated with TGc ELISA positivity may not be limitedto those presented here. Indeed, sera from patients with linearIgA dermatosis, herpes gestationis, vasculitis other thanWegener's granulomatosis, and IgA pemphigus also had raisedtiters in our human TGc ELISA; however, these data were notpresented as the number of patients in each group was less than10 and hence too small for statistical analysis. Moreover, arecent study [29] showed that 6.1% of patients having diversediagnoses but increased serum IgM rheumatoid factor levelswere false-positive in the TGc ELISA, suggesting that not onlyautoimmune diseases, but also certain serological conditionsmay be associated with increased ratio of false-positive results.
Other investigators failed to find increased anti-TGc IgA inchildren and young adults with type I diabetes mellitus [5] or in
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patients with ulcerative colitis and Crohn's disease [5,30] usinga guinea pig TGc ELISA system.
The increased total serum IgA Ab levels contributed to thefalse-positive TGc ELISA results, as shown by their statisticallysignificant correlation and the beneficial effect of raised NaClconcentration. This observation corresponds to previous find-ings [27], but it was not the sole reason for all false-positiveresults, because only one fifth of the tested AI sera had in-creased total IgA levels.
EMA-positive AI patients without GSD have only been veryrarely reported [31]. Our TGc ELISA positive AI patients werenegative for EMA, although it has been proven that IgA endo-mysial Abs recognize TGc epitopes [2,5,32,33]. Accordingly,the AI sera showed several bands in immunoblot studies notcorresponding to TGc. This confirms the presence of reactivityagainst minor contaminants in the purified preparation. Further-more, it is well known that IgA Abs in CD are directed againstconformation sensitive epitopes of TGc [2], thus it is not sur-prising that one of the CD patients' sera did not react with TGcin the blot; similarly, AI sera might have contained Abs directedagainst a number of proteins other than TGc that cannot bedetected by immunoblot.
The false positivity of most AI sera disappeared when ananti-TGc sandwich ELISA was produced binding only TGc tothe wells, indicating that reactivity to protein impurities was themajor factor responsible for the false-positive results. TheELISA based on the human TGc produced in bacteria furthersupported the involvement of protein contaminants in falsepositivity. This is consistent with other findings [25,26], but itshould be considered that our studies were performed using anon-commercial ELISA system, and that the high level of falsepositivity was only seen in a specially selected patient popula-tion not reflecting the clinical situation in which TGc ELISA isusually requested (the performance of our ELISAwas very goodregarding sera only from patients with gastrointestinal com-plaints). Thus the nature of our results cannot necessarily begeneralised to other (commercially available or home-made)TGc ELISAs, and they do not suggest a general low specificityof this diagnostic method. However, the identification of achaperone (heat shock protein 70) as a contaminant points to therisk of copurification of members of this class of proteins whenrecombinant ELISA antigens are purified by affinity chroma-tography. Specific analysis for the presence of such contami-nants should be considered.
Interestingly, the anti-TGc sandwich ELISA gave highlypositive signals for three sera of patients believed not to haveGSD, suggesting that some epitopes on the TGc molecule maybe hidden upon coating to an ELISA plate. This phenomenondraws attention to the limitations of the TGc ELISA, in favor ofdiagnostic systems in which serum IgA Abs react with unboundTGc [34].
In conclusion, the signal detected by our and a number ofother TGc ELISA systems in sera from patients with systemicAI disease appears to be related to technical problems (in oursystem, the presence of minor impurities and raised serum IgAAb levels) rather than the occurrence of TGc specific Abs inthese patients. Although several previous studies suggested a
possible association of anti-TGc Abs and certain conditionsindependent of gluten-sensitive disease, this notion could not beconfirmed by our work.
Acknowledgement
The authors thank Elke Dietzel, Petronella Sárdyné Izbéki,Birgitta Jakob, Ferencné Menyhárt, and Charlotte Ziesemer fortheir technical assistance, and Dr. Tamás Zágoni for providingsome patient sera. We are grateful to Dr. Róbert Király and Prof.László Fésüs for the kind gift of human TGc expressed inbacteria. The study was supported by the Deutsche Forschungs-gemeinschaft-Magyar Tudományos Akadémia (project 436UNG 113/135/0, Pa 660/2-1), the University Scientific Grant(ETT 155/2000) of Semmelweis University, and the Immun-diagnostik AG, Bensheim, Germany. M.S. was supported by afellowship of the Deutscher Akademischer Austauschdienst (A/98/23048), the Deutsche Forschungsgemeinschaft (FOR 265),the Protein Cross-Linking Programme of the European ScienceFoundation, the Friedrich and Maria Sophie Moritz Foundation,and the Maria Pesch Foundation. M.P., N.S., and Z.K. weresupported by the Deutsche Forschungsgemeinschaft (SFB 589).
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