Antibodies Against Synthetic Deamidated Gliadin Peptides and Tissue Transglutaminase for the Identification of Childhood Celiac Disease

CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2007;5:1276 1281 Antibodies Against Synthetic Deamidated Gliadin Peptides and Tissue Transglutaminase for the Identification of Childhood Celiac Disease DANIEL AGARDH Unit of Diabetes and Celiac Disease, Department of Clinical Sciences/Pediatrics, Lund University, Malmö, Sweden Background & Aims: Children with celiac disease have antibodies against gliadin, tissue transglutaminase (ttg), or both antigens. The aim was to evaluate immunoglobulin (Ig)A and IgG antibodies to synthetic deamidated gliadin peptides (DGP) and human ttg as screening markers for childhood celiac disease. Methods: Antibodies were detected in enzyme-linked immunosorbent assay using antihuman IgA, IgG, or a combined conjugate of IgA and IgG (IgAG) against DGP, ttg, or both (DGP/tTG), in sera from 119 children with celiac disease, 57 disease controls, and 398 blood donors. Treatment with a gluten-free diet was evaluated in 20 children with celiac disease who were followed up for 6 months from diagnosis. Results: The highest sensitivity was accounted for IgAG-DGP/tTG at 100% (119 of 119), followed by IgA-tTG at 97% (115 of 119), IgG-DGP at 95% (113 of 119), IgA-DGP at 91% (108 of 119), and IgG-tTG at 13% (15 of 119). With respect to disease controls and blood donors, specificity was for IgAG-DGP/ ttg at 89% (51 of 57) and at 97% (385 of 398), IgA-tTG at 96% (55 of 57) and at 98% (392 of 398), IgG-DGP at 86% (49 of 57) and at 99% (395 of 398), IgA-DGP at 91% (52 of 57) and at 92% (366 of 398), and IgG-tTG at 100%, respectively. The concordances between antibody assays were 87% 98%, except for comparisons with IgG-tTG (39% 41%). After 6 months of a gluten-free diet, the mean antibody levels decreased for all test results (P <.001). Conclusions: The combined IgAG-DGP/tTG assay is recommended as a frontline screening test for the identification of childhood celiac disease and also could be used as a marker of dietary compliance. Celiac disease (CD) is a common small-bowel disorder caused by permanent intolerance against gluten proteins in genetically susceptible individuals carrying the HLA- DQ2 (DQA1*0501-DQB1*02) and/or DQ8 (DQA1*0301- DQB1*0302) haplotypes. 1 Diagnosis is confirmed by intestinal biopsy specimens showing typical flattening of villous structure. 2 Treatment with a gluten-free diet leads to healing of the intestinal mucosa and the reintroduction of gluten results in a relapse of the disease. 3 CD is associated strongly with autoantibodies against tissue transglutaminase (ttg). This calcium-dependent enzyme belongs to a widely distributed group of enzymes involved in several important physiologic processes catalyzing posttranslational modification of proteins and peptides. 4 In CD, ttg is proposed to form new antigens of gluten peptides by deamidation of gliadin glutamines to glutamate that bind with high affinity to the HLA-DQ2 and DQ8 heterodimers. 5 Although ttg autoantibodies are highly specific markers for the disease, their role in its pathogenesis is still a matter of debate. 6 8 Antibodies are useful diagnostic tools in CD. A number of serologic tests already are available, of which antigliadin antibodies (AGA), endomysial autoantibodies (EMA), and ttg autoantibodies of the immunoglobulin (Ig)A isotype commonly are measured in clinical practice. It has been shown that the degree of intestinal damage also correlates with antibody levels. 9 Furthermore, antibodies are reduced after introduction of a gluten-free diet, which allows objective evaluation of how patients respond to treatment. 10 In children, the sensitivity and specificity of IgA-AGA has been estimated at 84% (52% 100%) and 91% (83% 100%), respectively. 11 25 The diagnostic performance of IgA-EMA reaches a sensitivity of 96% (88% 100%) and a specificity of 98% (90% 100%). 11,13 15,18,19,25 30 The expression of IgA-tTG shows a high correlation with IgA-EMA. 11 16 By using human recombinant ttg or native human ttg derived from red blood cells as the source of antigen in enzyme-linked immunosorbent assay (ELISA) kits, the diagnostic sensitivity of IgA-tTG is 96% (86% 100%) and the specificity is 97% (95% 100%). 13,17,18 As with IgA-EMA, the lower sensitivity of IgAtTG usually is the result of the inclusion of children younger than 2 years of age. 19 21 However, IgA antibodies are insufficient to detect CD in individuals with selective IgA deficiency; a disorder affecting approximately 1/500 of the general population and at a 10-fold increased risk for CD. 22 The estimated sensitivity and specificity of IgG-AGA in children is 93% (83% 100%) and 82% (65% 94%), respectively. 23 31 IgG-EMA yields both sensitivity and specificity near 100%, but studies mainly have been performed only on patients with IgA deficiency. 22,32,33 The diagnostic validity of IgG-tTG has, on the other hand, been shown to be reduced significantly by ELISA. 9,34 Recently, studies have documented very high sensitivity and specificity for detecting CD, with assays incorporating a fully synthetic, selectively deamidated peptide derived from whole native gliadin that incorporates multiple B-cell epitopes. 35 38 During the development of these novel deamidated gliadin peptide (DGP)-based assays, it was noticed that the specificity of Abbreviations used in this paper: AGA, antigliadin antibody; AU, arbitrary units; CD, celiac disease; DGP, deamidated gliadin peptide; ELISA, enzyme-linked immunosorbent assays; EMA, endomysial autoantibody; Ig, immunoglobulin; IgAG, combined conjugate of immunoglobulin A and immunoglobulin G; ttg, tissue transglutaminase. 2007 by the AGA Institute 1542-3565/07/$32.00 doi:10.1016/j.cgh.2007.05.024

November 2007 SEROLOGIC TESTS IN CELIAC DISEASE 1277 the IgG kit was close to 100%. This observation afforded the opportunity to use a conjugate that simultaneously detects both IgA and IgG antibodies. It also was observed that although the peptide-based assays had a high correlation with human ttg tests, there were still CD patients who had antibodies to DGP alone and some who reacted only to ttg. This observation led to the incorporation of DGP with human ttg in an attempt to develop a single, cost-effective assay that could be used to screen individuals for CD whether they are adult, pediatric, IgA sufficient, or IgA deficient. The aim of the present investigation was to evaluate a combined antibody assay using a conjugate of IgA and IgG (IgAG) against both synthetic DGP and human ttg. This dual conjugate is specific to the heavy chain of IgA and to the heavy chain of IgG blended together into a single reagent that detects both IgA and IgG. Our hypothesis was that this novel IgAG-DGP/tTG assay would improve the diagnostic accuracy in screening of childhood CD including infants and children with IgA deficiency. Patients and Methods Serum samples were obtained from 176 children with suspected CD who were admitted for an intestinal biopsy examination at the Department of Pediatrics, in Malmö. A total of 119 children (75 females, 44 males) had abnormal biopsy specimens 2 at a median of 5.7 years of age (range,.7 19.0 y) and were diagnosed with CD according to the revised criteria of the European Society of Pediatric Gastroenterology Hepatology and Nutrition. 3 The remaining 57 children (26 females, 31 males) had normal biopsy specimens at a median of 3.5 years of age (range,.9 14.6 y) and were considered to have disorders other than CD. In the disease control group, cow s milk protein intolerance or food allergy were diagnosed in 7 children, 5 had IgA deficiency, 3 had lipase deficiency, 2 had Helicobacter pylori gastritis, and 3 had transient EMA, of whom 1 had insulin-dependent diabetes mellitus. Another 4 children were investigated because of failure to thrive or short stature, and the remaining children had transient gastrointestinal symptoms. Included also were 87 CD children (57 females, 30 males) who were treated with a gluten-free diet for a median duration of 4.5 years (range,.5 16.5 y). All treated CD children had experienced relief of symptoms and showed clinical signs of remission on the gluten-free diet. Moreover, serum samples were taken from 20 children (13 females, 7 males) at diagnosis of CD at a median of 3.9 years (range, 1.3 13.9 y), and after the first 3 and 6 months of the gluten-free diet. As healthy controls, serum samples were collected from 398 adult blood donors (136 women, 262 men) at a median of 44 years of age (range, 19 81 y). Antibody Assays Six different kits from the QUANTA Lite series (INOVA Diagnostics, San Diego, CA) were tested: IgAG-DGP/tTG (QUANTA Lite h-ttg/dgp Screen), IgA-DGP (QUANTA Lite Gliadin IgA II), IgG-DGP (QUANTA Lite Gliadin IgG II), IgAG- DGP (QUANTA Lite Celiac DGP Screen), IgA-tTG (QUANTA Lite h-ttg IgA), and IgG-tTG (QUANTA Lite h-ttg IgG). These assays were run according to the manufacturer s instructions. Briefly, DGP and/or purified human erythrocyte ttg coated ELISA plates were incubated with diluted patient serum samples. Antibodies bound to the ELISA wells were detected with horseradish-peroxidase labeled anti-human IgA, IgG, or IgAG. Antibody levels were calculated from the optical density of the sample in relation to the reactivity of a positive control and expressed as arbitrary units (AU). Cut-off limits of less than 20 AU were defined as negative, 20 30 AU were defined as weakly positive, and greater than 30 AU were defined as moderate to strongly positive. Statistics Differences in antibody levels were tested using the Kruskal Wallis and Dunn s multiple comparison test. The Wilcoxon signed rank test was used to test significant change in autoantibody levels before and after the effect of the gluten-free diet. Correlations were evaluated using Spearman rank correlation (r) and P values less than.05 were considered significant. Results The diagnostic sensitivity and specificity of each assay is summarized in Table 1. By using the cut-off level set by the manufacturer of 20 AU for a positive result (Figure 1), the IgAG-DGP/tTG assay detected 119 of 119 (100%) untreated CD children. A total of 116 of 119 (97%) were positive for IgAG- DGP, 108 of 119 (91%) were positive for IgA-DGP, and 113 of 119 (95%) were positive for IgG-DGP. IgA-tTG detected 115 of 119 (97%), whereas only 15 of 119 (13%) were positive for IgG-tTG, including 1 of 2 children with IgA deficiency. The Table 1. Diagnostic Sensitivity and Specificity Untreated CD (n 119) Disease controls (n 57) Blood donors (n 398) ELISA Positive, n 2 n 1 Sensitivity, (%) Negative, n 1 n 2 Specificity, (%) Negative, n 1 n 2 Specificity, (%) IgAG-DGP/tTG 116 119 97.5 100 51 54 89.5 94.7 385 394 96.7 99.0 IgAG-DGP 110 116 92.4 97.5 51 56 89.5 98.2 380 388 95.5 97.5 IgA-DGP 95 108 79.8 90.8 52 54 91.2 94.7 366 385 92.0 96.7 IgG-DGP 105 113 88.2 95.0 49 56 86.0 98.2 395 396 99.2 99.5 IgA-tTG 113 115 95.0 96.6 55 57 96.5 100 392 396 98.5 99.5 IgG-tTG 5 15 4.2 12.6 57 100 398 100 NOTE. Two children with CD had IgA deficiency and were negative in IgA-DGP and IgA-tTG. n 1, Cut-off limit 20 AU defined as weak positive value; n 2, 30 AU defined as moderate to strong positive values.

1278 DANIEL AGARDH CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 5, No. 11 Figure 1. Antibody levels in children with (A) untreated CD (n 119), (B) treated CD (n 87), in (C) disease controls (n 57) and (D) adult blood donors (n 398) measured with 6 different ELISA kits: IgAG-DGP/tTG (I); IgAG- DGP (II); IgA-DGP (III); IgG-DGP (IV); IgAtTG (V); and IgG-tTG (VI). The dotted horizontal lines denote the cut-off level of upper normal. distribution of antibodies revealed that 12 of 119 (10%) were detected with all 6 assays, whereas 91 of 119 (76%) were positive for all antibodies except for IgG-tTG. The 2 untreated CD children with IgA deficiency were negative for both IgA-tTG and IgA-DGP, but positive in the other tests incorporating an anti- IgG conjugate. Two children were positive for IgAG-DGP/tTG in combination with IgA-tTG, and the remaining 12 had 3 or more antibodies. Of the 6 of 57 (11%) disease controls positive for IgAG-DGP/ ttg or IgAG-DGP, all had antibody levels less than 40 AU. IgA-DGP were found in 5 of 57 (9%) and IgG-DGP were found in 8 of 57 (14%) disease controls, whereas only 2 of 57 (4%) had IgA-tTG and none IgG-tTG. A total of 44 of 57 (77%) were negative for all ELISA tests, whereas 4 of 57 (7%) were positive for IgAG-DGP/tTG, IgAG-DGP, and IgG-DGP, and 4 of 57 (7%) were positive for IgG-DGP only. Two of 57 (4%) disease controls were positive for IgAG-DGP/tTG, IgG-DGP, and IgA-tTG, another 2 of 57 (4%) were positive for IgAG-DGP/tTG and IgA- DGP. One disease control with IgA deficiency was positive for IgG-tTG only. The concordance rates between antibody-positive and -negative children with untreated CD and disease controls are shown in Table 2. A total of 353 of 398 (89%) blood donors were negative in all 6 ELISA kits, whereas 21 of 398 (5%) were positive for IgA-DGP only, another 7 of 398 (2%) were positive for IgAG-DGP, and 4 of 398 (1%) were positive for both antibodies. In addition, 12 of 398 (3%) were positive for IgA-DGP in combination with one antibody or several other antibodies, and 1 individual was positive for IgG-DGP only. In children with treated CD, IgAG-DGP/tTG was positive in 22 of 87 (25%), 11 of 87 (12%) were positive for IgAG-DGP, IgG-DGP, or IgA-tTG, whereas 9 of 87 (10%) and 6 of 87 (7%) were positive for IgA-DGP and IgG-tTG, respectively. There was no correlation between antibody levels and the duration of the gluten-free diet for any of the ELISA tests, although the majority of antibody-positive children were found within the first 4-year period of gluten-free diet. The concordance between positive and negative results when IgAG-DGP/tTG was compared with IgAG-DGP was 74 of 87 (85%), 68 of 87 (78%) with IgA-DGP, 76 of 87 (87%) with IgG-DGP, 72 of 87 (83%) with IgA-tTG, and 71 of 87 (82%) with IgG-tTG. The response to the gluten-free diet in the 20 children with CD followed up for 6 months from diagnosis is shown in Figure 2. Discussion It is well known that the diagnostic sensitivity of EMA and ttg antibodies is reduced when testing very young children. Many or most of these EMA- and ttg-negative CD children are positive for antibodies to gliadin. As the disease progresses antibodies eventually are produced against both gliadin and ttg. Full expression of EMA and ttg antibodies Table 2. Concordance Between Positive and Negative Results in Children With CD and Disease Controls (n 176) Immunoassay IgAG-DGP/tTG IgAG-DGP IgA-DGP IgG-DGP IgA-tTG IgG-tTG IgAG-DGP/tTG 96.0% 87.5% 95.5% 95.5% 38.1% IgAG-DGP 96.0% 90.3% 94.9% 91.5% 39.2% IgA-DGP 87.5% 90.3% 85.2% 90.9% 41.5% IgG-DGP 95.5% 94.9% 85.2% 90.9% 39.8% IgA-tTG 95.5% 91.5% 90.9% 90.9% 40.9% IgG-tTG 38.1% 39.2% 41.5% 39.8% 40.9%

November 2007 SEROLOGIC TESTS IN CELIAC DISEASE 1279 Figure 2. Mean antibody levels in children with CD (n 20) at diagnosis and at 3 and 6 months of a gluten-free diet. Definition of ELISA kits: IgAG-DGP/tTG ( ); IgAG-DGP ( ); IgA-DGP ( ); IgG-DGP ( ); IgA-tTG ( ); IgG-tTG (Œ). *P.05; **P.001. generally occurs after 2 to 3 years of age. It therefore has been recommended to combine AGA with EMA or ttg antibodies to increase the diagnostic sensitivity of serologic testing during these earlier age intervals. 25,39,40 Still, conventional AGA assays also have performance problems, especially in the area of specificity, and reliance on these older assays has been discouraged. 11 24 As a consequence, many young children without CD often undergo invasive intestinal biopsy procedures and many CD children are not diagnosed because of a lack of reliable serologic markers. Previous studies on antibodies against synthetic DGP have proven to be more disease-specific and display much higher binding affinity for CD-associated antibodies compared with common AGA immunoassays. 36 38,41 In the present study, the DGP antigen was incorporated with several conformationally intact and deamidated B-cell epitopes derived from the gliadin. By using the cut-off level set by the manufacturer of 20 units for a positive result, 100% of the children in this study with untreated CD were detected by the IgAG-DGP/tTG assay including IgA-tTG negative children younger than 3 years of age, as well as those who had IgA deficiency. The corresponding specificity of this new assay was 89% with respect to disease controls and 97% with respect to blood donors. The specificity of the IgAG-DGP/tTG assay was less specific than that of IgA-tTG with respect to the disease controls (89% vs 96%), although the concordance of the 2 tests was high (96%). The specificities of the DGP assays obtained in this study are somewhat lower than reported previously for adult populations. 37,38 However, if the cut-off value is set at a level defined as moderate to strong positive ( 30 units), the specificity increases to 98% for disease controls and 99% for blood donors, and test sensitivity decreases to 97%. One possibility might be that the recommended cut-off values were nonoptimal for the pediatric population tested. An argument against altering test cut-off values is the fact that most disease controls and blood donors with a positive IgAG-DGP/tTG test also were found to be positive in one or more of the other assays as well. It therefore cannot be excluded that at least some of these individuals might have undiagnosed CD. It is particularly important to be able to distinguish patients with low from high dietary compliance and to objectively follow-up how the child responds to a gluten-free diet over time. IgA-DGP was the first antibody to decrease to normal levels during the first months of gluten-free diet. This indicates that the withdrawal of gluten is reflected first in the IgA antibody response against the DGP antigen. This response was not unexpected because CD probably is initiated in genetically susceptible individuals by deamidated fragments of wheat protein at or below mucosal surfaces. The eventual disappearance of ttg antibodies in the majority of CD patients after treatment with a gluten-free diet is supposed to be explained by the formation of covalent complexes between ttg and gliadin peptides via thioester bonds to the active site of the enzyme or via isopeptide bonds to lysine of ttg. 42 The novel ELISA assay described here derives its impressive sensitivity from 2 sources. It has been shown that assays incorporating either human ttg or the newer synthetic DGP perform well in a clinical setting with sensitivity and specificity for CD reported in the mid- to upper-90% range. By combining these 2 antigens on a single solid phase, the seronegative gap can be closed because untreated CD children negative for antibodies to one of the antigens may be positive for the other. Combination antigen assays also derive increased sensitivity by being able to detect those positive patients who may have low levels of antibody to both antigens just below the positive cut-off value of a single antigen test because of an additive effect. In the pediatric population described here there were indeed also active CD children who were negative for ttg antibodies showing up as positive for DGP individually and positive by the combined ttg/dgp screening test. Another source of the enhanced sensitivity shown by the IgAG-DGP/tTG assay is the use of a dual-isotype anti-human IgA and IgG conjugate. This dual conjugate makes it possible to detect IgA deficient as well as IgA sufficient CD patients. In our cohort, 2 of 119 untreated CD children showed IgA deficiency. These 2 patient samples were negative by IgAbased assays (both ttg and DGP) yet positive by the dualantigen dual-conjugate IgAG-DGP/tTG screening assay. This new screening test had no false-negative results and had a reasonable level of false-positive results. Adjustments of the cut-off value resulted in an optimal specificity. Alternatively, this new assay might be used as a sensitive screening tool to find potential CD in a rapid, cost-effective manner. The simplicity and efficiency of the test might allow more extensive serologic testing of at-risk but asymptomatic (or mild symptom) individuals. In conclusion, this study shows that children with CD have either IgA or IgG antibodies against DGP, ttg, or both antigens, of which antibody levels decrease in response to a glutenfree diet. The combined IgAG-DGP/tTG assay displays credible diagnostic accuracy and is recommended as a front-line screening test for the identification of childhood CD and could be used as a marker of dietary compliance after treatment with gluten-free diet. References 1. Sollid LM. Molecular basis of celiac disease. Annu Rev Immunol 2000;18:53 81.

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November 2007 SEROLOGIC TESTS IN CELIAC DISEASE 1281 41. Schwertz E, Kahlenberg F, Sack U, et al. Serologic assay based on gliadin-related nonapeptides as a highly sensitive and specific diagnostic aid in celiac disease. Clin Chem 2004;50:2370 2375. 42. Fleckenstein B, Qiao S, Larsen M, et al. Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides. J Biol Chem 2004;23:17607 17616. Address requests for reprints to: Daniel Agardh, Clinical Research Center, University Hospital MAS, SE-205 02 Malmö, Sweden. e-mail: daniel.agardh@med.lu.se; fax: (46) 40-391919. Supported by the Faculty of Medicine, Lund University, and the Skåne Council Foundation for Research Development, Malmö University Hospital. Walter Binder of INOVA Diagnostics, Inc., provided enzyme-linked immunosorbent assay kits for the study free of charge.