Anti-tissue Transglutaminase Enzyme-Linked Immunosorbent Assay for Screening of Celiac Disease in Moroccan Pediatric Patients

 

Younes Sbihi¹, Mohammed El Mzibri¹, Nezha Mouane², Abdelghani Iddar¹*

¹Biotechnology and Engineering of Biomolecules Unit, Division of Life Sciences,

National Centre for Nuclear Energy, Science and Technology (CNESTEN-Morocco), B.P 1382, Rabat, Morocco.

²Paediatrics’ Gastroenterology Hepatology and Nutrition Department - Academic Children’s Hospital Ibn Sina, Mohammed V University Rabat, Morocco.

 

ABSTRACT:

Celiac disease (CD) is an immune-mediated systemic disorder elicited by gluten which induces an enteropathy with atrophy of intestinal villi. No prevalence information was available for CD in Moroccan children because the diagnosis is based on multiple invasive duodenal biopsies. To simplify CD diagnosis, the objective of this work was to develop an Enzyme-linked Immunosorbent Assay (ELISA) for IgA and IgG tissue transglutaminase (tTG) antibodies detection suitable for Moroccan children. The concentration of the coated antigen and serum dilutions were optimized for the development of IgA anti- tTG and IgG anti-tTG ELISA detection in serum. The cut-off was calculated and the developed ELISAs were used for detecting IgA and IgG-tTG antibodies in 97 serum samples from 97 children aged 1 to 13 years, with variable clinical characteristics. After adjusting optimal working conditions of ELISA, the cut-offs were 0.04 and 0.156 AU for detecting anti-tTG and IgA anti-tTG IgG respectively. The use of IgG anti-tTG as CD marker (100 % of sensitivity) was better than the use of IgA anti-tTG in ELISA tests (90.3% sensitivity). In addition, the ELISA test shows a clear correlation between the IgG tTG antibodies titer and the degree of duodenal damage, estimated by the Marsh stages classification in patients with confirmed CD. Our results suggest that the assessment of the adapted IgG anti-tTG ELISA test can be used for CD in initial screening in Moroccan children after validation with a larger number of samples with CD for confirmation.

 

 

INTRODUCTION:

Celiac disease (CD), recognized as a public health problem, is an autoimmune enteropathy induced by gluten ingestion in genetically susceptible individuals. The ingestion of gluten by CD individuals causes chronic inflammations of the small intestinal mucosa and atrophy of intestinal villi with gastrointestinal-related symptoms (diarrhea and steatorrhea), fatigue, weight loss due to macro and micronutrients malabsorption, anemia and failure to thrive^1-3.

 

The main determinants of genetic susceptibility are HLA-DQA1 and HLA-DQB1 genes encoding for HLA-DQ2 and HLA-DQ8 molecules.

 

Selective deamidation of gliadin peptides by the ubiquitous intracellular enzyme, tissue transglutaminase (tTG), enhances their binding in the HLA DQ2 and DQ8 molecules⁴. TG may form covalent complexes with gliadin peptides resulting in the activation of gliadin-specific CD4 + lymphocytes. This reaction leads to the production of the collated anti-gliadin antibodies and anti-TG antibodies⁵.

 

CD is one of the most common diseases with a higher prevalence in the general population, often with vague non gastrointestinal symptoms^6,7. The prevalence of CD at any age varies greatly across different countries and shows a prevalence of 1/200 to 1/100 in the general population and up to 1/40 in patients with atypical presentations⁸. Studies conducted in children reported a prevalence of a ca. 1/100, in some European Nordic countries⁹. This prevalence is increasing in developing countries, particularly in the Middle East and North Africa where it is currently a common disorder¹⁰. In Morocco, as it’s the case of the other Maghreb countries, including Algeria, Tunisia, Libya and Egypt, a very high prevalence of CD has been reported in the general population^11-13. These high frequencies are associated with the consumption of wheat and barley as the major staple foods¹².

 

Inflammatory changes in the small intestinal villi, exposed to the highest concentration of gluten, make possible the diagnosis of CD. Villous atrophy can be scored by using Marsh classification. Normal mucosa intraepithelial lymphocytes were defined as Marsh stage I, infiltrative / hyperplastic lesions as Marsh stage II and villous atrophy corresponding to a stage III¹⁴. Early diagnosis and gluten-free diet may reduce risk of small bowel lymphoma^15,16. Multiple duodenal biopsies are generally recommended for histological evaluation to determine the insufficient tissue risk^17,18. This clinical practice is changing and the diagnosis is increasingly based on other parameters such as clinical signs, elevated serological markers (such as antibodies against TG, the enzyme responsible for the deamidation of gliadin peptides in intestinal lamina propria), in addition to pathological changes in a small-bowel biopsy¹⁹. Biopsies can be incredibly invasive and painful especially for children, and for these reasons, the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) has published a series of criteria to simplify CD diagnosis by avoiding small bowel biopsy in selected patients with clear symptoms, TG antibodies levels 10-fold above the upper limit of normal and positive anti-endomysial antibodies (IgA-EMA)^20,21. Of particular interest, sensitivity and specificity for CD greater than 95% has been described for IgA-EMA and IgA TG antibodies^10,22. Furthermore, higher titers of anti-TG in children have always been associated with histopathologic features of CD such as villous atrophy^23-25. The presence of specific autoantibodies reflecting the immunological response to gluten was performed by Enzyme Linked Immunosorbent Assay (ELISA) or radio ligand binding assay (RLA) by monitoring IgA class (the most sensitive antibodies) and also IgG which are mainly carried out in cases of IgA deficiency⁹. The approach using anti-TG dosages, due to their low cost, high reliability and feasibility as well as because they are not requiring expensive endoscopy and pathology equipment could facilitate the control CD epidemic and can reduce the risk of diagnosis errors in less privileged countries^25,26.

 

The present study was planned locally to develop Enzyme Linked Immunosorbent Assay (ELISA) for monitoring TG antibodies, to be used in coeliac disease diagnosis for a better management of this disease in Morocco.

 

MATERIALS AND METHODS:

Ethical approval:

The study protocol was approved by the Ethics Committee for Biomedical Research, Faculty of Medicine and Pharmacy of Rabat - Morocco, and written informed consent was obtained from each parent of the recruited patient.

 

Reagents:

Recombinant human tissue transglutaminase was purchased from Celikey™ Pharmacia Diagnostics. Bovine serum albumin (BSA), rabbit anti-human IgG –peroxidase conjugate, goat anti-human IgA –peroxidase conjugate, o-phenylenediamine dihydrochloride (OPD), hydrogen peroxide and polyoxyethylene-sorbitan monolaurate (Tween 20) were from Sigma-Aldrich (Merck KGaA). Nunc MaxiSorp microtiter plates were from Fisher Scientific. All the other products were of analytical reagent grade and were purchased from Fluka, Sigma-Aldrich, or Reidel-de Haein.

 

Patients’ blood samples:

Blood samples used in this study were obtained from 97 children, aged 1 to 13 years, with variable clinical characteristics. Thirty one (31) of them were confirmed as celiac by endoscopy and intestinal biopsy (7 patients had Marsh III grade, 11 had Marsh II grade and another 13 had Marsh I grade according to Marsh classification 1992)¹⁴. Fifty six (56) have other gastrointestinal disorders and teen (10) patients without any gastrointestinal disorders. These patients had been examined at the Pediatric Service in Children's Hospital Ibn Sina- Rabat-Morocco. The serum was obtained by centrifugation of blood samples at 5000 rpm for 10 min and stored at -20°C until examined using the developed ELISA. A total of 10 sera from patients with confirmed CD and 10 control sera from patients without any gastrointestinal disorders were used for the optimization of the experimental conditions and cut-offs calculation. Optimal experimental conditions were used to assess IgA-tTG and IgG-tTG statuses of the recruited patients.

 

Development and procedure of ELISA for IgA-tTG and IgG-tTG antibodies detection:

The ELISA was developed based on the protocol described by Stern et al. 2000 and Teesalu et al 2009 with some slight modifications^27,28, that were performed to optimize the ELISA testing procedures for IgA anti-tTG and IgG anti-tTG monitoring, including the concentration of the coated antigen (recombinant human tissue tTG) and serum dilutions. For this, microtiter plates were coated with 100µl of tTG (200 to 800ng /well) in Tris-HCl buffer (50mM, pH 7.5) containing NaCl (150mM), CaCl₂ (5mM) and NaN₃ (0.05%). The plates were washed with Tris-Buffered Saline-Tween-EDTA (TBS-TE: 50mM Tris–HCl at pH 7.5, 150mM NaCl, 1ml/l Tween 20, 10mM EDTA) and blocked by incubation with 0.5% BSA in TBS-TE containing 0.05% NaN₃ (300μl/well) overnight at 4°C. After three washing steps performed using TBS-TE supplemented with sucrose (5%), glycerol (10%) and NaN₃ (0.05%), the plates were dried at 25°C for 3 h, then sealed and stored at 4°C with desiccant  until use.

 

For serum dilutions optimization, 100μl/well of serum previously diluted with TBS-TE (1/1000 to 1/10) was crossed with concentrations of the coated tTG at 25°C for 2 h. The plates were exhaustively washed (4 times) with TBS-TE and 100 μl/well of a diluted (1/1000 in TBS-TE) goat anti-human IgA–peroxidase conjugate or rabbit anti-human IgG –peroxidase conjugate were added and incubated at 25°C for 1 hour. After another washing step for 3 times with TBS-TE followed by a washing step with distilled water, 200µl/well of O-phenylenediamine dihydro-chloride solution (0.4 mg/ml of OPD dissolved in 100 mM citrate-phosphate buffer, pH 5 containing 0.04% H2O2) was added. After incubation in the dark at room temperature for 30 min, the reaction was stopped by adding 50 µl/well of 3N HCl.  For each reaction, a negative control in which diluted serum template was omitted from the ELISA testing was included. The absorbance was read spectrophotometrically at 492 nm on an ELISA reader. The antibody concentrations were expressed in arbitrary units (AU). One arbitrary unit (1AU) is equivalent to the reciprocal serum dilution that gives an OD of 1 over background at 492 nm.

 

Cut-off determination:

The determination of an optimal cut-off value in ELISA assays has been estimated using independent negative sera of patients without CD.²⁹ Cut-off value was calculated by the following equation.

 

Cut - off = X + 3 × SD

 

Where X  is the mean and SD the standard deviation of independent negative control.

 

Statistical analyses:

The experimental data represent the mean of 3 independent assays. Student's-test was used; a p-value lower than 0.01 was considered significant. The sensitivity was expressed by the ratio of the true positives correctly identified by ELISA to the total of the true positives confirmed by the biopsy. The specificity is the proportion of true negatives identified by ELISA to the total of the true negatives confirmed by the biopsy.

 

RESULTS:

Optimization of ELISA for IgA anti-tTG and IgG anti-tTG detection:

In order to develop ELISA for detecting IgA anti-tTG and IgG anti-tTG, the amount of the recombinant tTG coating and the patient’s serum dilution were optimized using goat anti-human IgA or anti-human IgG conjugated to peroxidase. Various dilutions of sera (1/10 to 1/1000) were titrated against variety amounts of the coating antigens (200 to 800ng/well) to measure the reactivity of IgA-tTG antibodies and to select appropriate conditions of the test (Tables 1 and 2).

 

Table 1. Concentrations of coating and sera dilutions for IgG anti-tTG detection. 10 CD positive  and 10 negative without any gastrointestinal disorders sera were used for ELISA optimization

 

Table 2. Concentrations of coating and sera dilutions for IgA anti-tTG detection. 10 CD positive  and 10 negative without any gastrointestinal disorders sera were used for ELISA optimization

(NA) not applicable

 

The optical density (OD) using 10 positive sera (patients with CD confirmed by biopsy) and 10 negative control sera (10 patients without any gastrointestinal disorders) was determined at the different serum dilution. The cut-off was calculated using the mean and standard deviation according to the equation (1) using sera of for 10 healthy children. This cut-off was used to determine the positive and negative cases for the studied sera. For each condition of ELISA (antigen coating and serum dilution), the sensitivity was estimated. The conditions for detecting IgG anti-tTG with the best sensitivity were 400 ng/well coating, 1/500 sera dilution or 600 ng/well coating, 1/500 sera dilution (Table 1). For IgA anti-tTG, the best sensitivity was obtained with 600 ng/well coating and 1/100 sera dilution (Table 2).

 

Accordingly, optimal working conditions were 600 ng/well coating and 1/100 sera dilution with 0.04 AU as a cut-off for detecting IgA anti-tTG and, 400 ng/well coating and 1/500 sera dilution with 0.156 AU as a cut-off for detecting IgG anti-tTG. The protocols with optimal conditions were used for all subsequent experiments.

 

Serologic Data in Clinical Groups:

Figure 1 shows the serologic data of the 97 sera tested (31 with CD, 56 have other gastrointestinal disorders and 10 patients without any gastrointestinal disorders by ELISA systems using optimal working conditions. The cut-off values were determined by using 10 independent negative sera of patients. The preliminary cut-off value for the detection of IgG anti-tTG was 0.156 AU which was determined as the mean of 10 negative samples + (3 x standard deviation) (equation 1). The OD492 values of 32 samples were > 0.156 AU and 65 samples were below 0.156 AU (Figure 1). On the other hand, the cut-off value for detection of IgA anti-tTG was 0.040 AU. The OD492 values of 28 samples were > 0.040 AU and 69 negative samples were below 0.040 AU (Figure 1).

 

Optimal developed ELISA procedure was used to assess IgG anti-tTG and IgA anti-tTG status in sera from the 97 recruited patients. All the 31 confirmed-biopsy patients were positive samples using ELISA for IgG anti-tTG detection and apart for one false positive sample, 65 negative samples were confirmed by the developed test (Table 3).

 

Fig. 1. Serologic results in 97 sera by ELISA systems using optimal working conditions for detecting IgG anti-tTG and IgA anti-tTG. The cut-off was calculated using the mean and standard deviation using sera of for 10 healthy children; the Cut-off values are indicated by horizontal lines. Cut-offs for detection of IgG anti-tTG and IgA anti-tTG were 0.156 and 0.040 AU respectively.

 

Sensitivity and specificity:

For the detection of IgA anti-tTG, 28 out of 31 samples were tested positive with 3 false negative samples therefore, the total of negative samples was 69 (Table 3). Sensitivities and specificities based on the determined cut-off are shown in Table 3. The use of the ELISA for IgG anti-tTG detection exhibited 100% sensitivity and 98.5% specificity in the detection of CD. While the ELISA for IgA anti-tTG detection showed 90.3% sensitivity and 100% specificity.

 

Correlation of IgG anti-tTG in ELISA and Marsh grading in Patients with CD:

The assessment of IgG response intensity levels in ELISA with different grades of Marsh is shown in Table 4. Results showed an increasing trend of OD ranges from grade I to grade III since, total of 13 grade I patients had OD492 values in the range 0.156 to 0.601 AU, 11 grade II patients had OD492 values in the range 0.602 to 0.936 AU and 7  grade III patients with high OD492 values (0.937 to 1.652 AU). The Pearson product- moment correlation coefficient between the increasing OD492 and corresponding grade of disease was r = 0.899 with p <0.01. This results means there is positive correlation between IgG anti-tTG levels and progressive Marsh grading of duodenal biopsy in patients of Celiac disease.

 

Table 3. Comparison of sensitivities and specifies of IgG-tTG and IgA-tTG antibodies detection in the developed ELISA systems

 

 

 

Table 4. Marsh classification and IgG anti-tTG response intensity

 

DISCUSSION:

Genetic predisposition (Either one or both of the human leukocyte antigen DQ2 or DQ8 genes are carried by individuals with CD) and early introduction of wheat into children's diets have been reported to increase the risk of permanent intolerance gluten proteins³⁰. To the best of our knowledge, there’s no study addressing the prevalence of CD in Moroccan children. However, reported data showed that CD is frequent in North Africa, where high incidence of HLA-DQ2 haplotype and high consumption of gluten cereals were registered³¹. In addition, gluten cereals like wheat and barley are major staple foods of the most Moroccan population¹². Simplification of the diagnostic criteria and widespread use of serologic can help to estimate the true prevalence in populations and ensures a reliable and early diagnosis for a better management of the disease^32-34.

 

CD specific antibody tests are the initial tools that are used to identify individuals with probable CD. For these raisons, we developed in this study an in-house ELISA system for monitoring anti IgA and IgG-tTG antibodies.  It’s currently accepted that the ELISA is a precise and sensitive tool, widely used in clinical diagnostics to detect a wide range of diseases^35-37, and anti-TG antibodies are considered as the primary tools for screening candidates for duodenal biopsy and confirmation of CD diagnosis. Their sensitivity and specificity were higher to other serological tests such as anti-endomysial antibodies and anti-gliadin antibodies³⁸.

In the present study, the coating with the recombinant tTG and the dilution of serums were optimized. The optimal conditions were 600 ng/well coating and 1/100 sera dilution for detecting IgA anti-tTG and were 400 ng/well coating and 1/500 sera dilution for detecting IgG anti-tTG. In the most developed ELISAs, 0.5 or 1 µg were used as a standard amount of TG in coating and sera dilutions were 1/50 to 1/100 for IgA detection and were 1/100 to 1/500 for IgG detection^{27, 28}.

 

In our developed ELISA, the cut-off was calculated by adding three standard deviations to the mean optical density value of ELISA runs on the negative samples (healthy children). Several studies have used different cut-off titers for CD detection. The cut-off is generally computed by means of formulas that need absorbance readings of negative controls but when no controls are available, Receiver operated curve (ROC) curve was used to determine the cut-off for anti-TG detection²⁸. The cut-off values for a positive test may be different from one population to another depending on the diversity in gluten ingestion and genetic background. Therefore, there is a real need for the estimation of population-specific cut-off values of serological tests especially for anti-tTG.  Accordingly, and after optimization of the ELISA procedure, the cut-off values for detecting IgA anti-tTG and  IgG anti-tTG in Moroccan children were 0.04 AU and 0.156 AU, respectively.

 

These cut-offs were used for detecting IgA anti-tTG and IgG anti-tTG in 97 children sera.  For IgG anti-tTG ELISA, the titer values of 32 samples were > 0.156 AU and 65 samples were below 0.156 AU exhibiting 100% sensitivity and 98.5% specificity. For IgA anti-tTG ELISA,  the titer values of 82 samples were > 0.04 AU and 69 samples were below 0.04 AU exhibiting 90.3% sensitivity and 100% specificity in the detection of CD. The most serological tests for CD diagnostic based on anti-TG ELISA showed a great variability in terms of sensitivity and specificity, since the performance of the ELISA TG- antibody assays may vary depending on the quality and quantity of the TG antigen, the choice of coating buffers etc. and thus, may yield false-positive and false-negative results³⁹. Our results show that the use of IgG anti-tTG as CD markers in the collected blood children samples (100 % of sensitivity) was better than the use of IgA anti-tTG in ELISA tests. It is noted that previous studies have shown better diagnostic performance of IgA anti-tTG assays with sensitivity and specificity greater than 90% over IgG anti-tTG assays²⁸. IgG anti-tTG was only used for detecting CD patients with IgA-deficiency. The higher sensitivity can be explained by the fact that higher levels of IgG anti-tTG antibodies were detected in younger CD patients⁴⁰. Of particular interest, the ELISA test shows a clear correlation between the IgG tTG antibodies titer and the degree of duodenal damage, estimated by the Marsh stages in patients with confirmed CD. This correlation has been confirmed in several previous studies²⁰.

 

The present study is very informative and has much strength mainly related to the technical approach that was optimized to detect IgA anti-tTG and IgG anti-tTG with a specific characteristic of high sensitivity and specificity for IgG anti-tTG compared to IgA anti-tTG detection. However, the main limitation of the study is the low number of serum samples to validate the developed procedure. Moreover, and for more accuracy, this ELISA testing procedure must be validated by different laboratories on a larger number of blood children samples with CD for confirmation of the results and eventually standardization of the method. After that, anti-tTG ELISA can be suggested for initial screening of CD in Moroccan children as a relatively simple, non-invasive and less expensive method. We have developed an in-house immunoenzymatic assay adapted to the Moroccan population that could detect with high sensitivity and specificity serological biomarkers of CD for an optimal management of this disease.

 

CONFLICT OF INTEREST:

No potential conflict of interest was reported by the authors.

 

ACKNOWLEDGMENTS:

The authors would like to thank Dr. El Abbadi N, Moutaouakkil A, Dr. Moussaif A, Dr. Bouayyadi A, (CNESTEN, Rabat, Morocco) for their assistance and helpful suggestions

 

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Accepted on 05.04.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5436-5442.