Antibodies to tissue transglutaminase: comparison of ELISA and immunoprecipitation assay in the presence and in the absence of calcium ions

doi:10.1016/S0009-8981(00)00407-1

Clinica Chimica Acta

Volume 304, Issues 1–2, February 2001, Pages 75-84

https://doi.org/10.1016/S0009-8981(00)00407-1 Get rights and content

Abstract

Endomysial antibodies are characteristic of coeliac disease and tissue transglutaminase (tTG) has been identified as a major component of the endomysial antigen. tTG autoantibodies were measured in sera from patients with coeliac disease using ELISA based on guinea pig tTG and immunoprecipitation assay (IPA) based on ³⁵S-labelled human tTG produced in an in vitro transcription/translation system. In addition, the effect of calcium ions on the interaction between tTG autoantibodies in the two assays was studied. Under standard (i.e. Ca²⁺-free) conditions, 36/39 (92%) coeliac sera were positive for IgA tTG antibodies by ELISA and 34/39 (87%) sera were positive by IPA. Comparison of ELISA and IPA results showed three sera positive by ELISA but negative by IPA and one serum which was positive by IPA but negative by ELISA. Bland and Altman analysis of the correlation between the ELISA and IPA showed that the results for 37 out of 39 samples were in the agreement. The results by ELISA carried out without and with Ca²⁺were in good agreement (r=0.99; n=39). IPA using Ca²⁺containing buffer detected fewer samples compared to IPA using standard assay buffer however the results of the two assays also showed a good agreement (r=0.93; n=39). Our studies confirm that antibodies to tTG are good markers of coeliac disease and indicate that the autoantibody binding sites on tTG are formed in a way which is essentially independent of Ca²⁺.

Introduction

It is now estimated that autoimmune enteropathy characteristic of coeliac disease may affect in the region of 1 in 200 individuals [1], [2], [3]. Furthermore, coeliac disease tends to be associated with other autoimmune diseases, such as autoimmune thyroid disease (AITD), insulin dependent diabetes mellitus (IDDM), Sjorgen’s disease and Addison’s disease [1], [3]. Also, patients with selective IgA deficiency have an increased prevalence of coeliac disease [1], [3]. Endomysial antibody measured by a immunofluorescence test (IFT) has been shown to be a sensitive and specific marker for diagnosis and monitoring of coeliac disease [1], [2], [3]. Furthermore, tissue transglutaminase (tTG) has been identified as a major component of the endomysial antigen(s) involved in coeliac disease [4]. tTG structure and activity are markedly influenced by Ca²⁺ ions [5] and it has been suggested that a Ca²⁺ dependent conformation of tTG is important in forming tTG autoantibody binding sites [6], [7], [8]. Consequently, we have studied the effects of Ca²⁺ on the interaction between tTG autoantibodies and tTG of guinea pig and of human origin. Our results indicate that the autoantibody binding sites on tTG are formed in a way which is essentially independent of Ca²⁺.

Section snippets

Serum samples

Sera from 39 patients with coeliac disease (28 females, 11 males, median age 51, range 15–81 years) were used in the study. Diagnostic criteria in each patient were confirmed by histological examination of duodenal biopsy specimen which showed typical villous atrophy and clinical improvement with gluten free diet. Second biopsies were performed 9–12 months after initial diagnosis to confirm an improvement. Serum samples for this study were collected from patients prior to starting a gluten-free

Results

All patients had changes in their duodenal mucosa characteristic of coeliac disease as observed in biopsy specimens (Table 1). A total of 36 out of 39 (92%) sera from coeliac patients were positive for IgA tTG antibodies in the ELISA under standard (i.e. Ca²⁺ free) conditions. Twelve sera had levels of tTG antibodies between 10 and 100 units/ml, six sera had levels between 101 and 500 units/ml and eighteen sera had levels above 500 units/ml (Table 1). When the ELISA was carried out using Ca²⁺

Discussion

Our study showed that IgA tTG antibodies could be detected in 36/39 (92%) of coeliac patients using an ELISA based on guinea pig tTG preparations. This is in agreement with prevalences reported by other laboratories of 84–98% [4], [6], [7], [8], [12]. The results by ELISA carried out without and with Ca²⁺ were in a good agreement (Fig. 1), furthermore the same samples were positive in both assays. The two samples which were outside the±2S.D. range in the Bland and Altman analysis were highly

Acknowledgements

Ken Nakachi was in receipt of an RSR Fellowship. We are grateful to Dr. Shu Chen for help in carrying out statistical analysis and Miss C. Pegington and Miss C. James for their assistance in preparing the manuscript.

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In these experiments, radio-labeled rh-TG2 is expressed with a commercial kit based on a lysate of rabbit reticulocytes. Previous studies had indicated that calcium reduces the affinity between rh-TG2 and CD antibodies [43]. The explanation for this rather astonishing effect was the discovery of TG2 in the kit originating from the rabbit reticulocytes [44].
In 1997, a German group demonstrated that the antigen of the biomarker EMA (endomysial antibodies) in coeliac disease is a calcium-dependent thiol enzyme, transglutaminase type 2 (TG2). This most important discovery opened up an exciting field of research aimed at a better understanding of the pathogenesis of coeliac disease, a T-cell-driven autoimmune disorder with a prevalence of about 1%. The accidental activation of TG2, possibly caused by a stress-induced local deficiency of zinc in the intestinal wall, might play a key role where the enzyme catalyzes an atypical deamidation of specific glutamine residues of food gliadins. The genetic contribution is HLA DQ2 or DQ8, which can form a complex with the TG2-modified gliadin residues, resulting in an immune response with the formation of antibodies against both gliadin and the enzyme. Indeed, the immunopathogenesis of coeliac disease can now be recognized partly at the molecular level. Progress has already improved the opportunities for laboratory diagnostics and, hopefully, new ways of treating and preventing coeliac disease will become available. These exciting developments might stimulate research within other fields of autoimmune disorders. With its focus on TG2, this review highlights some of the intriguing mechanisms of the pathogenesis of coeliac disease, such as the structure of the neo-antigen, the involvement of calcium and zinc, and the effects of coeliac antibodies on TG2 activity. Moreover, the many pitfalls due to dubious laboratory practice are addressed, as is the potential when a fundamental biological mechanism is understood at the molecular level.
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Conflicting data have been published concerning the effect of calcium on binding of autoantibodies to tissue transglutaminase (tTG) in celiac disease (CD).
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Calcium increased binding of IgA-tTG and IgG-tTG in the ELISA test. The reverse effect observed in RBA may be explained by competitive binding between calcium activated native rabbit reticulocyte tTG and hr ³⁵S-tTG. tTG autoantibody assays may need taking calcium into account for accurate diagnostic sensitivity and specificity for CD.
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The interaction between IgA tissue transglutaminase (tTG) antibodies (Abs) and ³⁵S-labelled tTG produced in a transcription/translation (TnT) system with various amino acid (aa) deletions has been studied. These experiments showed that the tTG N-terminal aa 1–89 were important for tTG Ab binding in all 15 coeliac disease sera studied and the central residues (aa 401–491) were important for binding of tTG Abs in all but one sera. The contribution of C-terminal residues to tTG Ab binding varied in different coeliac sera but overall was less than the contributions of the N terminal and central regions.
Mouse monoclonal antibodies (MAbs) to tTG were produced and the tTG aa sequences recognised by the MAbs determined using modified ³⁵S-labelled tTG proteins. Analysis of the inhibiting effects of patient sera tTG Ab on binding of tTG MAbs to tTG confirmed the importance of the N-terminal and central regions of tTG in forming serum tTG Ab binding sites.
Recombinant human tTG was expressed in yeast and purified to better than 95% homogeneity using MAb affinity chromatography as a final purification step. This material was highly suitable for use in an ELISA for tTGAb.
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The description of a range of antibodies associated with coeliac disease has been an important development in the ability to test for this common and treatable condition non-invasively. However, the detection of these antibodies remains unstandardised and the appreciation of the clinical utility of each is evolving. In view of the advances in the diagnosis and understanding of coeliac disease, we discuss: (1) the relative advantages, disadvantages and comparative diagnostic utility of the different antibody tests including the confounding effect of selective IgA deficiency; (2) various technical aspects of these tests; (3) HLA-DQ typing as a supplementary tool to antibody testing; (4) areas of controversy resulting from insufficient or conflicting published data; and (5) potential testing strategies.
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2009, FEBS Journal
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2006, Journal of Endocrinological Investigation

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Antibodies to tissue transglutaminase: comparison of ELISA and immunoprecipitation assay in the presence and in the absence of calcium ions

Abstract

Introduction

Section snippets

Serum samples

Results

Discussion

Acknowledgements

The Lancet

J. Pediatr

Gastroenterology

Gastroenterology

Clin Chim. Acta

J. Biol. Chem.

J. Autoimmun.

Lancet

J. Immunol Methods

J. Chromatogr. B

J. Autoimmun.

J Immun Methods

Coeliac disease

Br. Med. J.

The structural basis for the regulation of tissue transglutaminase by calcium ions

Eur. J. Biochem.