Q J Med 2001; 94: 195-205
© 2001 Association of Physicians
Serum IgA tissue transglutaminase antibodies in coeliac disease and other gastrointestinal diseases
From the Gastrointestinal Unit, Department of Medical Sciences, University of Edinburgh, Western General Hospital, Edinburgh, UK
Received 21 January 2000 and in revised form 12 February 2001
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We investigated the presence of IgA anti-tissue transglutaminase (tTG) antibodies in untreated coeliac disease (CD) and other gastrointestinal diseases, and compared IgA tTG concentrations with anti-endomysium (EMA) immunofluorescent findings. The study included 116 untreated CD patients (74 female, 42 male, age range 15–78 years, median 47 years), 82 treated CD patients, 65 patients with normal duodenal histology, 260 disease control samples and 29 healthy volunteers. IgA anti-tTG, EMA, and anti-gliadin (AGA) antibodies were measured. Serum total IgA was measured in the CD patients. Two IgA-deficient untreated CD patients were excluded. IgA EMA and IgA AGA were positive in 99 (87%) and 69 (61%), respectively, of the 114 untreated CD patients. Elevated IgA anti-tTG were found in 92/114 (81%) untreated coeliacs, 1/82 (1%) treated coeliacs, 2/65 (3%) non-coeliacs, 10/260 (4%) disease controls and 2/29 (7%) volunteers. Four of the untreated CD patients, with a normal serum total IgA concentration, were negative for all the serological tests. IgA anti-tTG concentrations were significantly higher in untreated coeliacs (median 10200 units/ml) than in other groups (Mann–Whitney, p<0.00001) and compared well with IgA EMA titres (r2=0.54; p<0.0001).
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Introduction |
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Serological testing was included in the 1989 revision of the European Society of Paediatric Gastroenterology and Nutrition criteria for the diagnosis of coeliac disease (CD).1 Problems with the current serological methods include a lack of standardized methodologies, differing diagnostic practices and the varying populations studied. The sensitivities/specificities of IgA anti-gliadin antibodies (AGA) and IgA anti-endomysium antibodies (EMA) vary widely: 46–100%/86–100% for AGA,2–4 and 74–100%/91–100%5–8 for EMA. The recent finding that the enzyme tissue transglutaminase (tTG) is the auto-antigen that interacts with EMA antibodies in CD sera9 has led to the development of an enzyme-linked immunosorbent assay (ELISA). In theory, a semi-quantitative, objective ELISA would be superior to the labour-intensive, subjective, immunofluorescent EMA technique for population screening.
tTG is a calcium-dependent enzyme that catalyses the formation of 
-(
-glutamyl) lysine bonds between protein-bound glutamine and lysine residues, and is widely distributed in body fluids and tissues10 including the gastrointestinal submucosa.11,12 Through its cross-linking activity, tTG has been implicated in many physiological processes, including wound healing, via its interaction with extracellular matrix proteins.13 Gliadin is an excellent substrate for the enzyme, as it consists of about 40% glutamine residues,14 a necessary pre-requisite for the enzyme.10 In CD, tTG activity is increased in the jejunal mucosa.14 tTG provides a plausible mechanism for gliadin toxicity through its selective deamidation of glutamine residues of gliadin, creating epitopes which favour gliadin recognition by gut-derived T cells when presented by DQ2 molecules.15
The performance of the IgA anti-tTG ELISA has previously been reported in untreated CD patients with predominantly positive IgA EMA antibodies.16–19 This study differs from other recent reports in evaluating the IgA anti-tTG ELISA in diagnosing untreated CD patients, including a large proportion of IgA-EMA-negative CD patients. We have also compared the sensitivity of IgA anti-tTG to the conventionally used IgA EMA and IgA AGA tests. As tTG is widely distributed in the gut, we have also investigated its disease specificity by analysing sera from patients with a wide range of gastrointestinal disorders, including inflammatory, infectious, functional and neoplastic aetiologies. In addition, we address the issue of calcium activation of the enzyme on the assay characteristics, as there have been conflicting reports in recent studies, with some reporting calcium to be essential,16,17 while others disagree.18,20
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Patients
All patients recruited presented to the Gastroenterology Unit at the Western General Hospital in Edinburgh. Small-bowel mucosal biopsies were performed on clinical suspicion, and not on the basis of positive serology alone. Clinical presentations for which intestinal biopsy was performed included the investigation of diarrhoea, weight loss, anaemia, or the presence of an associated disorder (e.g. dermatitis herpetiformis or insulin-dependent diabetes mellitus). A total of 658 samples from 643 individuals were analysed (Table 1
). Ethical approval was obtained from the Medicine and Oncology Subcommittee of Lothian Ethics in Research Committee.
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Untreated CD patients
Serum samples were studied from 116 untreated CD patients (74 females, 42 males; age range 15–78 years, median 47 years) diagnosed between 1988 and 1999. Small-intestinal biopsies were obtained from all patients. Serum samples stored at -70 °C were available for all patients while on a normal gluten-containing diet and in 32 patients following gluten withdrawal (range 2–12months, median 5months). All untreated CD patients had characteristic histological findings and responded clinically to gluten withdrawal. IgA-EMA-negative CD patients were offered a repeat biopsy following treatment with a gluten-free diet (GFD) to support the diagnosis of CD.
Treated CD patients
Serum samples were analysed from 82 treated CD patients (62 females, 20 males; age range 15–82 years, median 53 years) attending the GI unit coeliac clinic. Dietary compliance was based on a dietary history and a negative IgA EMA antibody result.
Non-coeliac controls
Sera were obtained from 65 patients (45 females, 20 males; age range 16–90 years, median 45 years) investigated for suspected CD, but who subsequently had normal duodenal mucosal histology.
Disease controls
A total of 260 serum samples were analysed from 245 patients undergoing GI investigations. The diagnoses of these patients were Crohn's disease (68), ulcerative colitis (80), irritable bowel syndrome (36), infective or idiopathic diarrhoea (15) and miscellaneous other diagnoses including gastrointestinal malignancy and liver diseases (46). Small-bowel mucosal biopsies were not available for all of these patients.
Healthy controls
Serum samples were prospectively analysed from 29 healthy volunteers and 106 anonymous blood donors.
Laboratory methods
All serum samples were analysed for IgA-class AGA, EMA and anti-tTG antibodies. IgG AGA analyses were done in all of the untreated CD and non-CD control sera. IgA EMA antibodies were titrated to the end-point in 38 of the untreated CD patients. Serum total IgA was measured in all untreated CD patients. ELISA methods for determining AGA and total IgA were as previously described.21 The upper reference limits of IgA and IgG AGA were 
30 units/ml and 45 units/ml, respectively.
IgA anti-endomysium antibodies
Cryostat sections of human umbilical cord (8 microns thick) were cut and stored at -20 °C. Frozen sections were thawed at room temperature for 30 min, fixed in acetone for 10 min at -20 °C, and then transferred to chloroform for 30 min at room temperature. Each section was covered with diluent (0.9% sodium chloride, 0.05% Tween 20 and 1% adult bovine serum) for 10 min and then drained. Serum (50 µl) (diluted at 1/5) was added to each section and incubated at room temperature for 60 min. Each batch included a positive and a negative serum control.
Sections were rinsed in PBS, then FITC-conjugated sheep anti-human IgA (Scottish Antibody Production Unit, Product No. S073-201) (diluted at 1/20 in ELISA diluent) was added to each section, and they were incubated for 60 min in the dark. Slides were placed into a dish of PBS, rinsed with running water for 5 min, dried and then mounted in glycerin. The slides were examined by two independent readers at x10 and x25 magnifications using an immunofluorescence microscope (H500, Hund, Wetzlar), and a positive result was recorded if the connective tissue surrounding the muscle cells fluoresced brightly in a honeycomb pattern. The EMA was reported as equivocal if the readers could not confidently interpret the immunofluoresent pattern; this result did not represent disagreements between readers. The equivocal results have been grouped with the EMA-negative results for analysis in this paper. The two experienced readers were both blinded as to the final diagnoses and clinical details of the patients to avoid bias in this subjective assessment.
IgA anti-tissue transglutaminase antibodies
Immulon 2 (Dynatech) plates were coated with 100 ml/well of 10 mg/ml solution of guinea pig liver transglutaminase (Sigma, T5398) diluted in a carbonate-bicarbonate-coating buffer (0.05 M, pH 9.6) and incubated overnight in a covered moist box at 4 °C. Plates were washed three times with wash solution (0.9% saline, 0.05% Tween 20) and blocked with 250 ml/well of blocking solution (phosphate-buffered saline (PBS), 1% bovine serum albumin and 0.05% Tween 20) at room temperature for 2 h.
Serum from an untreated CD patient with known high anti-tTG antibody levels was used as a standard. The standard material was doubly diluted in duplicate from 1/50. High and low serum quality controls, and test serum samples were diluted in duplicate at 1/50 and 1/100 in a diluent of PBS with 0.05% Tween 20. Plates were incubated overnight at 4 °C and then washed three times in wash solution. Goat anti-human IgA (
-chain-specific) alkaline phosphatase conjugate, 100 ml/well, diluted at 1/800, was incubated at room temperature for 5 h and then washed three times with wash solution.
A p-nitrophenylphosphate substrate in diethanolamine buffer (Sigma) was added, and colour development was monitored by measuring A405 on an ELISA reader. The absorbance endpoint for IgA anti-tTG was taken when the top standard dilution reached an optical density of 1.0. Test results were technically acceptable if the quality control IgA anti-tTG concentrations were within ±2 SD of the mean of values obtained from previous runs, and the duplicate values differed by no more than 10%. The inter-assay and intra-assay coefficients of variation were 9.1% and 11.7%, respectively. The serum reference standard was designated as 25000 units/ml and the lower limit of detection in serum was 300 units/ml. The normal upper reference range for the IgA anti-tTG ELISA of 2950 units/ml was calculated using a non-parametric percentile method22 which excluded 95% of the 106 anonymous blood donors.
Effect of calcium and pH during the coating stage of the tTG ELISA
To investigate the requirement for calcium during the coating stage, we added 5 mM calcium chloride (Sigma) to the carbonate-bicarbonate coating buffer (pH 9.6) and compared this to a Tris/HCl system (pH 7.5) with 5 mM calcium chloride. Doubling dilutions of three serum samples were performed and A405 readings were recorded. BSA in the blocking solution was substituted with haemoglobin at 1 mg/ml to exclude possible interaction of antibodies with BSA.
Statistical analysis
The sensitivity of the IgA anti-tTG ELISA was calculated as the frequency of positive results in biopsy-proven CD patients while on a normal diet. The specificity of the assay was calculated from the group of non-CD controls found to have normal duodenal histology, and was not based on the disease controls from whom an intestinal biopsy was not obtained. The Mann–Whitney test was used to compare IgA anti-tTG antibody concentrations in untreated CD patients and the disease control groups. Pearson's correlation was applied to determine the correlation between serum IgA anti-tTG and the corresponding IgA EMA titre, and the significance was determined by linear regression.
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Effect of calcium during the coating stage of the tTG ELISA
The addition of 5 mM calcium chloride to the carbonate-bicarbonate buffer (pH 9.6) resulted in a reduction of optical density readings in two of the three samples studied. The optical densities recorded using the carbonate-bicarbonate system without calcium were more comparable to the Tris/HCl coating method (pH of 7.5) with 5 mmol/l of calcium than following the addition of calcium to our system (Figure 1
).
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Serum total IgA in untreated coeliac disease patients
Two of the 116 untreated CD patients had selective IgA deficiency (total serum IgA <0.07 mg/l). Duodenal histology had improved markedly in both of these patients following treatment with a GFD. Serum IgA-class anti-tTG antibodies, AGA and EMA were not detectable. IgG AGA was raised in both patients. IgG anti-tTG antibodies were not measured. IgA-class serology results from these two male patients have been excluded from the calculations of assay sensitivity (n=114).
Serum IgA anti-tTG antibody and IgA EMA
Untreated coeliac disease patients
IgA anti-tTG antibodies were raised in 81% (92/114) of the untreated CD patients and 1% (1/82) of the treated CD patients (Table 2). Serum IgA anti-tTG antibody concentrations were significantly higher in untreated CD patients (median 10 200 units/ml; range 300–89 400 units/ml) than in all other control groups (Mann–Whitney test, p<0.0001) (Figure 2). Of the 22 untreated CD patients with normal serum IgA anti-tTG and total IgA concentrations, 11 were positive for IgA EMA, six for IgA AGA and 14 for IgG AGA (Table 3). Three untreated CD patients had borderline negative IgA anti-tTG concentrations of 2220 units/ml, 2340 units/ml and 2510 units/ml, respectively.
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IgA EMA was positive in 99/114 (87%) of untreated CD patients, negative in 14/114 (12%) and equivocal in 1/114 (1%). The IgA anti-tTG antibody concentration was raised in four of these patients. The IgA AGA and IgG AGA were raised in three and 11 of these patients, respectively. Repeat biopsy following treatment with a GFD was done in 12/15 EMA-negative CD patients. Duodenal histology had improved in nine, but remained unchanged in four, of whom two were non-compliant with their diet. The two patients who did not have repeat duodenal biopsies both had a clinical response to gluten withdrawal.
The sensitivity of the IgA anti-tTG ELISA would increase from 81% (92/114) to 89% (88/99) if only the IgA-EMA-positive patients were included. Using a combination of tests could further enhance the serological detection of untreated CD (Table 4). The highest sensitivities in diagnosing CD of 93% and 96% resulted from the combination with IgG AGA of either IgA anti-tTG or IgA EMA, respectively. The use of IgG AGA as a screening test, however, produces a false-positive rate in controls of 22% (14/65). The combination of IgA anti-tTG or IgA EMA with IgA AGA produced sensitivities of 86% and 89%, respectively, with a lower false-positive detection rate for IgA AGA of 9/65 (14%).
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Control patients
Elevated IgA anti-tTG results were found in 1/82 (1%) of treated CD patients, in 2/65 (3%) of non-CD controls, in 10/260 (4%) of gastrointestinal disease controls and in 2/29 (7%) of healthy volunteers (Table 2
). Of the treated CD patients, 14/82 (17%) remained IgA AGA-positive. One of the two non-CD controls with an elevated IgA anti-tTG concentration of 29 000 units/ml, but negative IgA EMA, was being investigated for diarrhoea and abdominal cramps and had a normal duodenal biopsy. In retrospect, this lady admitted to consuming a low gluten diet at the time of her intestinal biopsy and serum sampling. This patient declined a supervised gluten challenge and repeat small-intestinal biopsy. The other non-CD control with a raised IgA anti-tTG concentration of 4860 units/ml, was a 40-year-old lady who presented with symptoms of abdominal discomfort, loose stools alternating with constipation and dyspepsia. An upper endoscopy with duodenal histology and a barium enema were both normal. The IgA and IgG AGA were negative. Her symptoms were thought to be due to functional bowel disease.
One Crohn's disease patient with an IgA anti-tTG concentration of 5260 units/ml had a positive IgA EMA but a normal duodenal biopsy including intra-epithelial lymphocyte count. One patient with ulcerative colitis had a raised IgA anti-tTG concentration of 18 100 units/ml but a negative IgA EMA and AGA. This individual has not undergone small-intestinal biopsy.
Two of the healthy male volunteers had elevated IgA anti-tTG concentrations of 6495 units/ml and 3315 units/ml, respectively. Only the former was EMA positive and subsequent duodenal biopsies showed partial villous atrophy consistent with untreated CD. In retrospect, this ‘asymptomatic’ member of the medical staff reported intermittent diarrhoea and dyspepsia. There was no family history of CD, but his sister is currently being investigated for anaemia. The other volunteer was IgA AGA- and EMA-negative and has not undergone duodenal biopsy.
Other serological results
Serum IgA and IgG AGA were raised in 61% (69/114) and 87% (99/114) of untreated CD patients, respectively, and in 14% (9/65) and 22% (14/65) of non-CD controls with normal duodenal biopsies, respectively. Elevated IgA AGA concentrations were also seen in 15% (39/260) of disease controls and 17% (14/82) of treated CD patients, but in none of the 29 healthy volunteers. Four untreated CD patients were negative for all serological markers studied.
IgA anti-tTG antibodies following treatment with a gluten-free diet
Of the 116 untreated CD patients, serum samples were obtained from 32 after treatment with a gluten free diet (GFD) for a median 5months (range 2–12months). IgA anti-tTG titres were elevated in 23/32 (72%) patients before commencing the GFD. IgA anti-tTG antibody concentrations remained negative following gluten withdrawal in nine (28%) patients and are not shown. IgA anti-tTG antibody concentrations reduced rapidly following GFD introduction (usually within the first 5months), returning to normal in 18/23 (78%) of patients within 12months (IgA anti-tTG concentrations pre-GFD: median 15 700 U/ml, range 4480–81 600 U/ml; post-GFD: median 1621 U/ml, range 613–7304 units/ml) (Figure 3). Two patients consented to attend regularly for close monitoring of IgA anti-tTG concentrations after starting of their GFD and the decline in their antibody concentrations is shown in Figure 4. IgA AGA and IgA EMA antibodies were positive in 22/32 (69%) and in 24/32 (75%) untreated CD patients, respectively. Of the 24 EMA-positive patients, 15 (47%) sero-reverted, compared to six (27%) of the 22 IgA AGA-positive patients during the study period.
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Association between serum IgA anti-tTG and EMA antibodies
Serum IgA anti-tTG antibodies were significantly higher in the 101 EMA-positive (median 12 600 units/ml; range <300–89 400 U/ml) than in the 449 EMA-negative (median 620 units/ml; range 300–18 100 units/ml) serum samples (Mann–Whitney test, p<0.0001) (Figure 5). The IgA anti-tTG concentrations in 37 IgA EMA-positive untreated CD patients correlated well with the IgA EMA titres (r=0.54; p<0.0001). The IgA anti-tTG was not significantly higher in patients with partial villous atrophy (PVA) compared to those with increased intra-epithelial lymphocytes (IELs), or in those with subtotal villous atrophy (STVA) compared to those with PVA (Mann–Whitney, p=0.3 and p=0.7, respectively) (Figure 6).
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Discussion |
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Serological testing using the IgA anti-tTG ELISA is useful, but is not superior to the IgA EMA immunofluorescent test in screening for CD. IgA anti-tTG concentrations are higher in untreated CD than in treated CD, other gastrointestinal diseases, or healthy controls. The sensitivities of the IgA anti-tTG ELISA and IgA EMA were comparable at 81% and 87%, respectively, and IgA anti-tTG concentrations and IgA EMA titres compared well. Our finding of positive IgA anti-tTG and EMA serology in a healthy ‘asymptomatic’ volunteer with partial villous atrophy supports the observation that subtle presentations of CD may easily be missed.23,24
Past reports of assay sensitivity and specificity have been greatly influenced by performing intestinal biopsy on predominantly seropositive patients.25 The CD patients in this study however, were biopsied on the basis of clinical suspicion, and were not selected exclusively by serology. This approach produced a high prevalence of EMA-negative untreated CD patients (15%, 17/116), of whom only two had selective IgA deficiency. The other subject had an equivocal EMA result. The high proportion of EMA-negative CD patients reduced the IgA anti-tTG ELISA sensitivity, as it improved from 81% to 89% when only EMA-positive CD patients were considered. The sensitivities of IgA anti-tTG and EMA improved when used in combination with IgG AGA, giving sensitivities of 93% and 96%, respectively. The addition of IgG AGA, however, resulted in a reduction in specificity.
The IgA anti-tTG ELISA positively identified four EMA-negative untreated CD patients, but failed to detect 11 EMA-positive patients. Others have also found discrepancies between IgA anti-tTG and EMA.17 We found positive IgA anti-tTG, IgA EMA and IgA AGA antibodies in 4% (10/260), 0.4% (1/260) and 15% (39/260) of our disease controls respectively. Sulkanen et al. did not find positive IgA anti-tTG antibodies in any of their 32 IBD patients, but did find positive results in other gastrointestinal controls.16 The finding of IgA anti-tTG antibodies in non-CD subjects may be explained by the wide distribution of the enzyme throughout the gastrointestinal tract.11,12 The lower specificity of the IgA anti-tTG ELISA than the IgA EMA may also be due to the use of a non-human, guinea pig liver as the source of tTG in comparison to the use of human umbilical cord, used in the EMA assay. The high IgA anti-tTG antibody titres found in untreated CD patients is probably due to the suitability of dietary gliadin as a substrate for tTG, and because the concentration of gliadin is highest in the proximal intestine.
Recent reports on the sensitivities and specificities of the IgA anti-tTG ELISA (also using the guinea pig liver source) have ranged from 85% to 98.1% and from 94% to 98%, respectively.16–19,26 However, few of the untreated CD patients investigated in these studies were EMA-negative: 10/136,16 1/106,17 1/27,18 and 0/39.19 Rostami et al. found that the sensitivities of IgA anti-tTG27 and IgA EMA28 correlated closely with histological severity. EMA positivity has also been thought to depend on the length of intestine involved,29 as well as on genetic factors.30 However, we did not find an association between antibody titres and histological severity.
The requirement for calcium activation of the enzyme has been disputed. In our hands, the enhanced sensitivity of calcium activation appears to be pH-dependent, as in our assay (pH 9.6), the optical density readings fell sharply following the addition of calcium. The finding that calcium is not essential is in agreement with recent reports.18,20
Recently, a radio-binding assay using human recombinant tTG has been shown to compare well with EMA findings, with a sensitivity of 99.1% (111/112) and specificity of 95.7%.31 This technique was superior to the IgA anti- tTG ELISA using guinea pig liver tTG dissolved in PBS at pH 7.3 (also without calcium and blocked with BSA), which had a sensitivity of 85% and specificity of 91% in the same patient group.31 These latter findings compare well with our results in EMA-positive patients using a similar method except for the coating pH. Human recombinant tTG would appear to be better than the guinea pig liver source, which only shares 80% sequence homology with the human enzyme. Both the sensitivity and specificity of the IgA anti-tTG ELISA may improve in the future when a human recombinant tTG becomes commercially available.
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Acknowledgments |
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Preliminary results of this study were presented as a poster at the British Society of Gastroenterology meeting in Glasgow, Scotland, in March 1999, and published as an abstract in Gut 1999; 44(suppl. 1): W299:A75. The study has also been presented in abstract and poster forms at the Changing Features of Coeliac Disease International Conference in Tampere, Finland, in June 1998, and at the Eighth International Symposium on Coeliac Disease in Naples, Italy, in April 1999. We thank John Bode and Kathleen Kingstone for their advice and technical contributions, and Pearl Culbert for her clerical assistance. We are grateful to the Scottish Office for funding this project which is dedicated to the memory of the late Professor Anne Ferguson.
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Address correspondence to Dr S. Ghosh, Gastrointestinal Unit, Western General Hospital, Edinburgh EH4 2XU. sg{at}srv\|[oslash]\|.med.ed.ac.uk
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References |
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