Antibodies to tissue transglutaminase: comparison of ELISA and immunoprecipitation assay in the presence and in the absence of calcium ions
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+.
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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.
References (29)
- et al.
Coeliac disease
The Lancet
(1997) Tissue transglutaminase: the Holy Grail for the diagnosis of celiac disease, at last?
J. Pediatr
(1999)- et al.
Autoantibodies to tissue transglutaminase as predictors of celiac disease
Gastroenterology
(1998) - et al.
Tissue transglutaminase autoantibody enzyme-linked immunosorbent assay in detecting celiac disease
Gastroenterology
(1998) - et al.
Correlation between tissue transglutaminase antibodies and endomysium antibodies as diagnostic markers of coeliac disease
Clin Chim. Acta
(1999) - et al.
Isolation and characterisation of cDNA clones to mouse macrophage and human endothelial cell tissue transglutaminase
J. Biol. Chem.
(1991) - et al.
Comparison of tissue transglutaminase-specific antibody assays with established antibody measurements for coeliac disease
J. Autoimmun.
(1999) - et al.
Tissue transglutaminase and combined screening for coeliac disease and type 1 diabetes-associated autoantibodies
Lancet
(1998) - et al.
Evaluation of a high-throughput second antibody radiobinding assay for measuring IgA antibodies to human tissue transglutaminase
J. Immunol Methods
(1999) - et al.
Transglutaminases: purification and activity assays
J. Chromatogr. B
(1996)
One third of H.L.A. DQ2 homozygous patients with type 1 diabetes express coeliac disease-associated transglutaminase autoantibodies
J. Autoimmun.
Evaluation of a high-throughput second antibody radiobinding assay for measuring IgA antibodies to human tissue transglutaminase
J Immun Methods
Coeliac disease
Br. Med. J.
The structural basis for the regulation of tissue transglutaminase by calcium ions
Eur. J. Biochem.
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