Immunochromatographic sticks for tissue transglutaminase and antigliadin antibody screening in celiac disease

CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2004;2:480 – 484

Immunochromatographic Sticks for Tissue Transglutaminase and Antigliadin Antibody Screening in Celiac Disease ´ PEZ,* CARMEN RIBES– KONINCKX,‡ CARLOS GENZOR,§ SUSANA GAMEN,§ SERGIO FERRE–LO ¶ ˜ A, LUIS ORTIGOSA,㛳 and ENRIQUE ME´NDEZ* LUIS PEN *Unidad de Gluten, Centro Nacional de Biotecnologı´a, Cantoblanco, Madrid; ‡Unidad de Gastroenterologı´a Infantil, Hospital Universitario La Fe, Valencia; §Operon S.A., Cuarte de Huerva, Zaragoza; ¶Departamento de Pediatrı´a, Hospital Universitario Materno-Infantil de Canarias, Las Palmas De Gran Canaria; and 㛳Departamento de Pediatrı´a, Unidad de Gastroenterologı´a Infantil, Hospital Universitario de la Candelaria, Santa Cruz de Tenerife, Spain

Background & Aims: We investigated two 1-step immunochromatographic visual assays based on human recombinant tissue-transglutaminase (t-TG) as an alternative to enzyme-linked immunosorbent assays (ELISAs) for celiac disease (CD) screening. Methods: We used a tissue-transglutaminase (t-TG) stick, which detected immunoglobulin A/G (IgA/G) antibodies to t-TG, and a t-TG/antigliadin antibodies (AGA) stick, which detected IgA antibodies to both t-TG and AGA, as well as t-TG and AGA ELISAs, to determine t-TG and AGA antibodies in untreated celiac patients with subtotal villous atrophy. A total of 142 children (3 IgA-deficient sera) and 30 adults, and 140 controls (normal mucosa; 121 children and 19 adults), plus 23 sera from pediatric CD patients in remission were assayed. Results: For pediatric patients, with the t-TG stick we obtained a sensitivity of 97.1% and a specificity of 99.0%, and in adults, 83.3% and 100%, respectively. The t-TG/AGA stick displayed a sensitivity of 95.7% and a specificity of 99.0% for t-TG and a sensitivity of 89.2% and a specificity of 95.8% for AGA in children, and a sensitivity of 80% and a specificity of 100% for t-TG and a sensitivity of 83.3% and a specificity of 100% for AGA in adults. Results were comparable with the corresponding ELISAs. Conclusions: The 2 visual assays are efficient for CD screening as an alternative to ELISAs. They are simple to handle and to interpret. By the combined use of the 2 sticks, IgAdeficient patients can be identified as positive in the t-TG (IgG/A) but negative in the t-TG/AGA (IgA) stick.

he high prevalence of celiac disease (CD), about 1 in 160,1–5 together with mortality (neoplasias) and morbidity (osteopenia and autoimmune diseases) associated with untreated CD,6 –9 argue for early diagnosis and treatment. Because histologic studies of the mucosa are necessary to confirm enteropathy,10 highly sensitive and specific serologic markers (i.e., antigliadin antibodies [AGA] and antiendomysial antibodies) are used to select candidates for small intestinal biopsy examination.11–15 Since the publication of reports identifying tissue-transglutaminase (t-TG) as the major autoantigen in endomy-

T

sial structures,16 t-TG enzyme-linked immunosorbent assay (ELISA) has become the most universally accepted technique for CD screening.17–20 However, ELISA and immunofluorescence methods require well-equipped laboratories, a trained staff, and are not available worldwide. Despite the interest in cheap and easy-to-perform tests, up until now only a few such assays have been reported: our visual immunoassay based on gliadin,21 a dot-blot based on human recombinant t-TG (Rt-TG),22 and a more recent immunochromatographic strip using guinea pig t-TG.23 We report a new generation of visual immunochromatographic methods for CD screening based on human Rt-TG: the t-TG stick for immunoglobulin A/G (IgA/G) antibodies to t-TG, and the t-TG/ antigliadin antibodies (AGA) stick for both IgA antibodies to t-TG and to AGA.

Methods Immunochromatographic Sticks The immunochromatographic stick assays we have developed use lateral flow strips of 2 different kinds (Operon, Zaragoza, Spain), which consist of several layers of materials, including all necessary reagents.

t-TG Stick The t-TG stick is a 2-line test. Sticks are dipped in sera, 1:20 diluted in phosphate-buffered saline containing 1% bovine serum albumin, and placed into eppendorf (Hamburg, Germany) tubes or microtiter plate wells. If a sample contains anti–t-TG antibodies, liquid rises vertically by capillarity and reconstitutes dried red microspheres coated with Rt-TG placed on an absorbent pad. The resuspended red microsphere/Rt-TG/anti–t-TG complex reaches a Abbreviations used in this paper: AGA, antigliadin antibodies; CD, celiac disease; ELISA, enzyme-linked immunosorbent assay; Ig, immunoglobulin; Rt-TG, recombinant human tissue-transglutaminase; t-TG, tissue-transglutaminase; UCD, untreated celiac disease. © 2004 by the American Gastroenterological Association 1542-3565/04/$30.00 PII: 10.1053/S1542-3565(04)00166-1

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ethanolic extract was concentrated by ultrafiltration and the gliadin was lyophilized. Anti-human IgA monoclonal antibody was produced by Operon.

ELISA Tests

Figure 1. Immunochromatographic stick display of positive and negative CD sera. The first 3 columns correspond to t-TG sticks and the remaining columns correspond to t-TG/AGA sticks.

line of immobilized Rt-TG, giving an immunoreaction resulting in a red band. The red line indicates the presence of IgA and/or IgG class anti–t-TG antibodies in the sample. As a control system of the assay, blue microspheres coated with a protein also are placed on the absorbent pad. The resuspended blue microsphere/protein complex reaches a line of immobilized antibodies against this protein, resulting in a blue band. If no blue line appears, the test is considered to be invalid. In the absence of anti–t-TG antibody in the sample, no red line appears, only a blue line (Figure 1).

t-TG/AGA Stick The t-TG/AGA stick is a 3-line test. Samples are diluted 1:20 in a buffered solution containing bovine serum albumin plus a nonionic detergent and placed into eppendorf tubes or microtiter plates wells. After dipping the stick into a diluted serum, if it contains IgA anti–t-TG and/or AGAs, dried red microspheres coated with a monoclonal antibody against human IgA, located on an absorbent pad, are reconstituted. The resuspended red microsphere/monoclonal antibody anti-human IgA/IgA antigliadin or IgA anti–t-TG complex ascends vertically and eventually reacts with separated immobilized lines containing either purified gliadins or RtTG, resulting in 1 or 2 red bands at different levels. The sticks also include a blue control line, as described earlier. The blue control line appears at the top; the red AGA line, indicating IgA reactivity against gliadins, appears in the middle; and the red t-TG line, indicating IgA anti–t-TG, appears at the bottom of the strips (Figure 1). Both the t-TG and t-TG/AGA stick tests take only 5–10 minutes to perform. Reading the test results of the assays was performed at appearance of the blue control lines and no later than 20 minutes after the test was performed. It was performed blindly by at least 2 independent observers, and results were expressed as crosses and graded as 1⫹ to 3⫹. The developed lines are stable for weeks, either at room temperature or refrigerated. Human Rt-TG was obtained from Diarect (Friburg, Germany); the gliadin was extracted from a variety of wheat.24 The

IgA/AGA were measured by using either a homemade ELISA method24 or the CAP gliadin IgA fluorometric enzyme immunoassay (FEIA) (Pharmacia & Upjohn, Freiburg, Germany), taking into consideration the cut-off levels of each method for accurate evaluation of results. All serum samples also were tested for IgA t-TG antibodies by a commercial assay (Celikey; Pharmacia & Upjohn, Uppsala, Sweden), applying a cut-off level of 6 IU/mL. Total serum IgA levels also were available from all patients.

Controls and Patients A total of 335 consecutive sera, 286 from pediatric patients, age range 0.9 –13 years, and 49 from adults, aged 16– 65 years, were included. For one serum to be selected for the study, an intestinal biopsy examination must have been performed within 1 month either before or after the serum sampling, and no dietary restrictions had been imposed on the patients. Retrospective evaluation of the study population established that 142 children and 30 adults had a severe intestinal villous atrophy at serum sampling, and a diagnosis of CD had been confirmed according to international criteria25 in all of them. They are referred to as the untreated CD patients (UCD) as shown in Table 1. In 121 children and 19 adults with normal or minor histologic changes of the mucosa, other gastrointestinal diseases other than CD had been established, with the most frequent diagnosis in the pediatric group being cow’s milk protein intolerance, giardiasis, and postenteritis syndrome (Table 1). The remaining 23 sera pertained to 23 children with CD, aged 5.5–7.5 years, on a gluten-free diet for at least 2 years (treated CD) and showing normal or minor histologic changes in their mucosa (Table 1).

Results As shown in Table 2, there is a very high correlation between the different visual assays and the respective ELISAs as far as the pediatric population is concerned. Table 1. Groups of Patients Assayed in the Study

Untreated CDa Treated CDc,d Other gastrointestinal diseasesd Total number aSevere

Children (age range, 0.9–13 yr)

Adults (age range, 16–65 yr)

142b 23 121 286

30 — 19 49

villous atrophy of the intestinal mucosa. 3 IgA-deficient patients. cCeliac patients on gluten-free diet. dNormal mucosa or minor histological changes. bIncluding

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Table 2. Sensitivity and Specificity of the Sticks Versus ELISA in CD Screening in Pediatric Patients t-TG stick (IgA/G)

t-TG/AGA stick (IgA)

t-TG Band(IgA/G)

t-TG ELISA(IgA)

Band(IgA)

t-TG ⫹ AGAa

AGA ELISA(IgA)

Band(IgA)

ELISA(IgA)

Band(IgA)

ELISA(IgA)

CDb

Untreated (139)c Positive tests Negative tests Control groupd (121) Positive tests Negative tests Sensitivity (%) Specificity (%) Concordance (%) Total number of sera

135 4

134 5

133 6

134 5

124 15

120 19

138 1

137 2

2 119 97.1 99.0

2 119 96.4 99.0

2 119 95.7 99.0

2 119 96.4 99.0

5 116 89.2 95.8

13 108 86.3 89.2

6 115 99.3 95.0

15 106 98.6 87.6

99.3 260c

99.3 260c

96.7 260c

99.3 260c

aOnly

one visual test vs. 2 ELISA tests. villous atrophy. cThe 3 IgA-deficient sera have been excluded. dNormal mucosa or minor histologic changes. bSevere

Three of the 142 pediatric UCD patients had selective IgA deficiency and are not included in the statistical analysis (Table 2). They showed a positive band in the t-TG stick but no bands in the IgA t-TG/AGA assay. They also were negative in the IgA ELISA assays. Sensitivity and specificity results for CD diagnosis obtained for the 139 remaining UCD children and for the 121 controls (non-CD) for the t-TG stick and the t-TG/AGA stick are practically identical to those of the corresponding ELISAs (Table 2). With the t-TG stick for IgA/G t-TG antibody detection, we obtained a sensitivity of 97.1% and a specificity of 99.0% for CD diagnosis, with a concordance of 99.3% with the t-TG ELISA. The t-TG/AGA stick displayed a sensitivity of 95.7% and a specificity of 99.0% for t-TG and a sensitivity of 89.2% and a specificity of 95.8% for AGA, with a concordance of 99.3% and 96.7% with the corresponding t-TG and AGA ELISA. Four sera from UCD children were found to be t-TG negative both in the t-TG stick and in the ELISA, and one more was negative only in the IgA t-TG ELISA. Two more sera displayed no t-TG band in the t-TG/AGA stick. In the non-CD group, 2 children gave a positive band in the t-TG stick and a low positive result in the t-TG ELISA. Three of the 23 patients with treated CD displayed a weak positive band in the t-TG stick and a low positive result, just above the reference value, in the t-TG ELISA. They had a normal mucosa, although dietary transgressions cannot be ruled out entirely. They tested negative with the IgA t-TG/AGA stick, thus indicating that t-TG antibodies probably were mostly IgG. By using the t-TG/AGA stick, and regarding the appearance of at least one serologic marker as positive, we

obtained a very high sensitivity for CD diagnosis (i.e., 99.3%). This is even higher than that obtained by combining 2 ELISAs, one for IgA t-TG along with one for AGA, which yielded a sensitivity of 98.6%. The positive predictive value for CD diagnosis of at least one positive serologic marker, either t-TG or AGA, is 95.8% for the t-TG/AGA stick and 90.0% for the 2 ELISAs. The negativity of both markers has a negative predictive value of 99.1% and 98.1%, respectively, with an overall efficiency for CD diagnosis of 97.3% and 93.4%, respectively. As for the adult patients, 5 sera from the UCD group were negative in the t-TG stick, 4 of them being also negative in the ELISA and 1 with t-TG just above the reference value. On the contrary, one patient with no IgA t-TG as determined by ELISA displayed a weak positive band in the t-TG stick. As shown in Table 3, we obtained a sensitivity of 83.3% and a specificity of 100% for the t-TG (IgA/G) stick, corresponding values for the ELISA IgA t-TG being 83.3% and 94.7%, respectively.

Discussion One of the advantages of these stick systems, as compared with previously published visual methods, is the use of Rt-TG as the antigen, which has been shown in previous studies to increase the efficiency of the anti– t-TG assays.20,26 Selective IgA deficiency adds complexity to serologic CD screening.27 The t-TG stick detecting IgG as well as IgA anti–t-TG antibodies, total serum IgA level determination, as a previous step toward correct interpretation of IgA class serologic markers, is not essential.

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Table 3. Sensitivity and Specificity of the Sticks Versus ELISA in CD Screening in Adult Patients t-TG stick (IgA/G)

t-TG/AGA stick (IgA)

t-TG Band(IgA/G)

t-TG ELISA(IgA)

Band(IgA)

t-TG ⫹ AGAa

AGA ELISA(IgA)

Band(IgA)

ELISA(IgA)

Band(IgA)

ELISA(IgA)

CDb

Untreated (30) Positive tests Negative tests Control groupc (19) Positive tests Negative tests Sensitivity (%) Specificity (%) Concordance (%) Total number of sera

25 5

25 5

0 19 83.3 100

1 18 83.3 94.7

24 6

25 5

0 19 80 100

93.3 49

1 18 83.3 94.7 92 49

25 5

27 3

0 19 83.3 100

2 17 90 89.4 80 49

26 4

27 3

0 19 86.6 100

3 16 90 84.3 83.7 49

aOnly

one visual test vs. 2 ELISA tests. villous atrophy. cNormal mucosa or minor histologic changes. bSevere

In the pediatric group, the IgA/G t-TG stick assay displays a very high sensitivity and specificity, similar to that obtained for the ELISA t-TG assays both in our study population as well as in previous published studies.2,7,13 As for the t-TG/AGA stick, sensitivity and specificity of the t-TG/AGA stick for CD diagnosis for each individual marker is similar to that obtained for AGA or t-TG by ELISA. Negativity of 2 sera from UCDs using the t-TG/AGA stick, when both have tested positive with the IgA/G t-TG stick, indicates that the antibodies are probably IgG. There are no clinical features that clearly distinguish t-TG–negative from t-TG–positive UCD patients. The combined use of IgA AGA plus t-TG antibody determination increases diagnostic sensitivity for CD screening. This sensitivity is slightly higher for the tTG/AGA stick than for the combined use of 2 independent ELISAs, 1 for IgA t-TG plus 1 for AGA. Thus, a quick and simple procedure can take the place of 2 more complex and time-consuming assays for CD screening. The control group included 121 children, matched for age with the UCD patients, who had cow’s milk protein intolerance, giardiasis, postenteritis syndrome, and so forth, conditions that are frequent in the pediatric population and difficult to discriminate from CD in clinical practice. For diagnostic purposes all children had small intestinal biopsy examinations performed, showing minor histologic lesions that did not suggest gluten-induced enteropathy. The high specificity for both visual assays confirms them as extremely valuable diagnostic tools in this age group. On the whole, the high positive and negative predictive value of the t-TG/AGA stick for CD diagnosis in the pediatric group is noteworthy. In the adult population, sensitivity for both visual stick assays is lower than for the pediatric population but

similar to that obtained for the corresponding ELISAs. Indeed, it has been shown that efficiency of serologic markers for CD diagnosis is lower in adults than in children.28,29 The fact that pediatric CD patients can respond to a different gliadin peptide repertoire compared with adults, thus enhancing a diversity in T-cell response,30 might account for the difference in immunologic response in the 2 different age groups. The high specificity obtained in the adult population both in the visual assays and in the ELISAs could be related to the low number of patients included, owing to the difficulty in recruiting adults with a normal small intestinal biopsy examination. Although the population tested was significantly larger than in a previously published report using guinea pig t-TG as an antigen with a similar visual method,23 we feel a broader study including populations with different prevalence and risk rates, currently underway, is needed to confirm the promising results obtained.

Conclusions An outstanding feature of these 2 immunochromatographic visual sticks is that they provide similar or even slightly higher sensitivity and specificity values than the corresponding t-TG ELISA or the AGA ELISA, and prove extremely efficient for CD screening. The combined t-TG/AGA stick detects both IgA antibodies to AGA and t-TG in a single assay (Figure 1), and would eliminate the need for 2 parallel IgA t-TG or AGA ELISA immunoassays and, in turn, result in a cost reduction. The entire assay requires no more than 5–10 minutes to perform, and is simple to handle and interpret, thus enabling a large number of sera to be processed in a short time, with no need for special equipment or a

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qualified operator. Preliminary data obtained in this study show that both visual methods are useful tools to screen for CD in large populations, to survey groups at risk for CD, and to select candidates for small intestinal biopsy examination. Moreover, the simplicity of the systems makes them especially appropriate for developing countries.

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Address requests for reprints to: E. Me ´ndez, Unidad de Gluten, Centro Nacional de Biotecnologı´a, Cantoblanco, Madrid, Spain. e-mail: [email protected]; fax: (34) 91-585-45-06. Supported by a grant from the Ministerio de Ciencia y Tecnologı´a (BIO2000-0403-P4-03), coordinated by the group of the Unidad de Gluten, Centro Nacional de Biotecnologı´a, and the Operon Company. Enrique Me ´ndez, Carlos Genzor, and Susana Gamen developed the sticks. Carmen Ribes-Koninckx, Luis Pen ˜a, and Luis Ortigosa selected the sera. Sergio Ferre-Lo ´pez, Carmen Ribes-Koninckx, Carlos Genzor, and Susana Gamen performed the stick assays and enzyme-linked immunosorbent assays. The assays performed by inventors of the device were independently confirmed by Dr. Carmen Ribes-Koninckx, M.D., Ph.D. The authors thank Jean Boyd for assistance with the English translation.