Anal Bioanal Chem (2012) 403:1191–1194 DOI 10.1007/s00216-011-5702-z

TECHNICAL NOTE

An ELIME assay for the rapid diagnosis of coeliac disease Gianluca Adornetto & Giulia Volpe & Alessia De Stefano & Sonia Martini & Giuseppina Gallucci & Angelo Manzoni & Sergio Bernardini & Marco Mascini & Danila Moscone

Received: 13 October 2011 / Revised: 22 December 2011 / Accepted: 29 December 2011 / Published online: 19 January 2012 # Springer-Verlag 2012

Abstract Coeliac disease (CD) is a gluten-induced autoimmune enteropathy found in genetically susceptible subjects. Because of the high number of undetected cases, rapid and cheaper screening methods are needed. Currently, the CD diagnosis involves the detection of anti-transglutaminase IgA antibodies (anti-tTG IgA) in blood serum through the use of ELISA systems with confirmation by histology of the intestinal mucosa. A new, rapid magneto-electrochemical Published in the special issue Euroanalysis XVI (The European Conference on Analytical Chemistry) with guest editor Slavica Ražić. Electronic supplementary material The online version of this article (doi:10.1007/s00216-011-5702-z) contains supplementary material, which is available to authorized users. G. Adornetto : G. Volpe : A. De Stefano : D. Moscone (*) Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy e-mail: [email protected] S. Martini : G. Gallucci Radim SpA, Via del Mare 125, 00040 Pomezia, RM, Italy A. Manzoni SEAC-RADIM Group—Research and Development Department, Via di Prato 74, 50041 Calenzano, Florence, Italy S. Bernardini Dipartimento di Medicina Interna, Università di Roma Tor Vergata, 00133 Rome, Italy M. Mascini Dipartimento di Chimica, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy

immunosensor for CD diagnosis has been developed and applied to serum sample analysis. The system uses magnetic beads coated with tTG antigen to detect anti-tTG antibodies in positive serum samples and an alkaline phosphataseconjugated anti-human IgA as label. An electrochemical readout, using magnetized screen-printed electrodes coupled with a portable instrument, is made after the addition of αnaphtyl phosphate, which is enzymatically converted into the electrochemically active α-naphthol product. The work involved the following considerations: (1) optimization of analytical parameters; (2) recovery evaluation, adding known concentrations of anti-tTG IgA to “blank” sera; (3) analysis of 107 blood serum samples; (4) calculation of the ROC curve, resulting in a cut-off of 1.0 U/ml, 100% of clinical sensitivity and 98.36% of clinical specificity; evaluation of the agreement between electrochemical and ELISA kit values (r2 of 0.943). The system developed could be an useful tool for a correct and rapid CD diagnosis. This method is simple, cheap, rapid, and suitable for screening analyses performed outside of the classical diagnostic laboratory. Keywords Coeliac disease . Transglutaminase . Magnetic beads . Electrochemical sensor

Introduction The coeliac disease (CD) is a chronic inflammatory intestinal disorder triggered by the ingestion of gluten (the protein fraction present in wheat, rye, and barley) in genetically susceptible individuals. It is considered an autoimmune disorder with a high prevalence in Europe and North America (1 affected every 100–300 individuals) [1, 2]. In subjects having particular HLA class II aplotypes (DQ2 or DQ8) [3], the ingestion of gluten causes a strong autoimmune response

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that leads to gastrointestinal symptoms (chronic diarrhea, abdominal distention and pain, weakness and malabsorption) as well as non gastrointestinal ones (dermatitis herpetiformis, anemia, etc.) [4]. Furthermore, untreated patients can develop other chronic or malignant diseases like intestinal lymphoma, esophageal carcinoma, osteoporosis, etc. [5, 6]. For these reasons, early diagnosis and treatment with a gluten-free diet are very important in reducing mortality and the occurrence of CD-associated disorders [7]. The histological demonstration of characteristic lesions of the intestinal mucosa remains the gold standard for diagnosing CD [8]. The use of serological markers, such as antitissue transglutaminase IgA autoantibodies (anti-tTG IgA), antibodies against gliadin, and endomysium are widely used for screening and selecting candidates for intestinal biopsy [8–11]. ELISA techniques are routinely used for the detection of these markers. Some authors [12] reported a radioimmunological assay, based on the measurement of anti-tTG IgA, as a useful test to monitor CD. Although this method is reliable and sensitive, it suffers from problems associated with the use of radioisotopes, the cost of scintillation fluids, radioactive waste disposal, and restriction of its use to institutes having the permission for handling radioisotopes. Because of an increased number of subclinical and undetected cases [13], the development of rapid, cheap, and simple screening methods, suitable for analysis carried out outside the classical diagnostic laboratory, is highly needed in order to prevent negative outcomes for those affected. To meet this need, in this work we describe a novel approach for measuring anti-tTG IgA antibodies in blood serum samples. The proposed method, an enzyme-linked immunomagnetic electrochemical (ELIME) assay [14], is based on the use of magnetic beads Fig. 1 Scheme and apparatus of the ELIME assay

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(MBs) as support for the immunological chain tTG/anti-tTG IgA/anti-human IgA-alkaline phosphatase (AP), coupled with an array of eight-magnetized screen-printed electrodes. The electroactive product of the enzymatic reaction (Fig. 1) is quickly measured. The procedure adopted involves the reaction, for 15 min, between a few microliters of tTG-coated magnetic beads and the specific antibodies (anti-tTG IgA) present in standards or in the patient’s sera. After washing, an aliquot of alkaline phosphatase-labeled anti-human IgA (IgAAP) is added and left to react for 10 min. Then, the MBs (with the immobilized immunological chain tTG/anti-tTG IgA/antihuman IgA-AP) are confined, with the aid of a magnet, onto the surface of the working electrode (WE) before the AP substrate, 1-naphthyl phosphate, is added and left to react for 2 min. The enzymatic product 1-naphtol is then measured using a rapid and sensitive electrochemical technique, the differential pulse voltammetry (for materials, methods and complete protocol, see the Electronic Supplementary Material). Our system shows the necessary conditions for the purposes of screening and diagnosis of celiac disease, i.e., high clinical sensitivity and specificity, as well as other features required for analysis carried out outside the classical diagnostic laboratory: short analysis time, simplicity of use and miniaturized size of the cheap portable apparatus (both instrument and disposable screen-printed electrodes—SPEs).

Results and discussion Preliminary experiments, aimed at developing a method to detect low concentrations of anti-tTG IgA, were carried out

An ELIME assay for the rapid diagnosis of coeliac disease

analyzing four different anti-tTG IgA calibrators (0, 8, 50, 100 U/ml) for each parameter under study. Some parameters, such as the volume of buffer for MBs resuspension (50 or 100 μl), the amounts of MBs transferred onto the WE surface (10 or 20 μl) and the volume of calibrators (15 or 30 μl) were carefully optimized. For each condition, a calibration curve was constructed by plotting the current signals (microamperes) against the different calibrator concentrations, and a linear response up to 50 arbitrary units (AU)/ml was obtained. The linear calibration curves were fitted by linear regression and a progressive increase of the analytical sensitivity (represented by the different slope values) was observed. In particular, the best conditions, selected for further experiments, were found utilizing 30 μl of calibrators, 50 μl of DEA buffer, and 20 μl of tTG-coated MBs. The calibration straight line [y0(0.040± 0.001) x+(0.028±0.041), r2 00.994] resulting with these latter conditions (Electronic Supplementary Material Figure S1, blue line) showed a fourfold increase of the slope with respect to that obtained in the first tests (0,009±0.001, performed using 15 μl of calibrators, 100 μl of DEA buffer and 10 μl of tTG-coated-MBs—Electronic Supplementary Material Figure S1, black line). The limit of detection (LOD) and the limit of quantification (LOQ) of the ELIME assay were calculated by analyzing for 20 times a human blank serum. LOD and LOQ, defined as the concentration values corresponding to the signal obtained by adding three (for LOD) or six (for LOQ) standard deviations of zero point (blank sample) to the mean value of the blank sample measurements (i.e. mean value+3× or 6× SD) were found to be 0.7 and 1 AU/ml, respectively. To assess the precision and recovery of the proposed method, three replicates of blank serum spiked with antitTG IgA antibodies at different concentrations (4, 10, 25, 50 AU/ml) were prepared and analyzed on each of 3 different days (n018 for each concentration level: 3 replicates× 3 days×2 electrodes). Precision was determined by calculating the CV for the replicate measurements and recovery (R%) was calculated as 100×(measured content/expected content). The results are shown in Table 1. Having set-up the system and evaluated of its analytical proprieties, the method was employed for blind analysis of 107 serum samples (61 negative plus 46 positives samples) obtained and separately tested by the University Hospital Tor Vergata, using an anti-tTG IgA spectrophotometric Table 1 Precision and recovery of the ELIME assay

Expected value (AU/ml) 4 10 25 50

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Fig. 2 Receiver operating characteristic (ROC curve) for 107 serum samples

ELISA kit and an anti-endomysial immunofluorescent assay [11] as confirmatory method. Each sample was analyzed a number of times (between three and five) on different days and the results of this study showed that our method was able to discriminate all the positive samples and 60 negative ones. Concerning positive samples, the endogenous amount of anti-tTG IgA, quantified by our ELIME assay, ranges from 2.5±22% to 3,300±5% AU/ml. In order to perform a diagnostic test evaluation, the results obtained were used to calculate a receiver operating characteristic (ROC) curve [15] (Fig. 2), using the statistic software MedCalc. The calculated cut-off (threshold value to discriminate positive samples from negative ones) of our method was found to be 1.0 AU/ml, with 98.36% of clinical specificity and 100% of clinical sensitivity. It is known that the discriminating capacity of a test is proportional to the extension area under the ROC curve (AUC, area under curve); in this context we obtained an AUC equal to 1.0, a result that classifies our test as “perfect”, according to the criteria proposed by Swets [16]. Finally, in order to assess the agreement between our method and the ELISA spectrophotometric KIT, a correlation curve was calculated using 26 serum samples, measured by both the proposed ELIME method and the ELISA kit. All the highly positive serum samples, with values higher than 50 AU/ml, were analyzed by ELIME after appropriate dilution, to make them fall in the linear range of the calibration

Mean value found ±SD (AU/ml)

R (%)

CV (%)

4±1 10±2 24±3 48±8

100 100 96 96

25 20 13 16

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curve. However, since the samples with values higher than 50 AU/ml were not quantified by the ELISA kit, but only classified as highly positive, the correlation line was constructed by plotting only the values of the 26 samples with an endogenous content of anti-tTG IgA quantified by our system between 1 and 50 AU/mL (upper limit of linearity for both ELISA and ELIME assay). The regression line equation was: y0(0.998±0.050)x−(2.855±0.996), r2 00.943. The intercept different from zero reveals a systematic error that may be attributed to a difference between the calibrators of two systems; in fact, international standards of IgA anti-tTG do not exist and all commercial kits express its own concentration values as arbitrary units (AU) and not as international units. In conclusion, this study strongly supports the ELIME antitransglutaminase autoantibody method as a sensitive and specific test for the screening of celiac disease. This test, when compared with the fluorimetric RAD analyzer, shows similar clinical specificity and sensitivity (respectively, 100% and 97% for RAD), the same analysis time and a lower cut-off (1.00 AU/ml and 6.00 AU/ml for ELIME and for RAD respectively). However, the fluorimetric RAD system employs a cumbersome and expensive instrumentation, suitable only for hospital laboratories, while the ELIME method is based on the use of a cheap, portable and miniaturized apparatus (Palmsens instrument dimensions, 15.5× 8.0× 3.0 cm; eight-sensors strip dimensions, 8.5×4.0×0.5 cm). Concerning the comparison between the ELIME assay and the spectrophotometric ELISA kit, we obtained similar clinical specificity and sensitivity values (98.57% and 100% for ELISA kit), a lower cut-off (7.00 AU/ml for ELISA kit), and a shorter analysis time (about 30 min for ELIME compared with 2 h for ELISA). Although the size of the commercially available ELISA detectors are highly variable, our electrochemical instrument is however less expensive, smaller and suitable for analysis performed outside the classic diagnostic laboratory. Moreover, because of the low detection limit and the high sensitivity of the ELIME assay, experiments are underway in our laboratory to apply the method to the analysis of the secretory anti-tTG IgA present in saliva. Acknowledgments The project, titled “Biosensori per la determinazione rapida (POCT) della celiachia e delle allergie.” has been funded by the Regione Lazio, in the Bando FILAS: Progetti di Ricerca Industriale e/o Sviluppo Sperimentale.

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References 1. Ciclitira PJ, King AL, Fraser JS (2001) AGA technical review on celiac sprue. American Gastroenterological Association. Gastroenterology 120:636–651 2. Maki M, Mustalahti K, Kokkonen J, Kulmala P, Haapalahti M, Karttunen T, Ilonen J, Laurila K, Dahlbom I, Hansson T, Hopfl P, Knip M (2003) Prevalence of celiac disease among children in Finland. N Engl J Med 348:2517–2524 3. Sollid L, Thorsby E (1993) HLA subsceptibility genes in celiac disease: genetic mapping and role in pathogenesis. Gastroenterology 105:910–922 4. Catassi C, Fabiani E (1997) The spectrum of coelic disease in children. Bailliere Clin Gastroenterol 11:485–507 5. Collin P, Reunala T, Pukkala E, Laippala P, Keyrilainen O, Pasternak A (1994) Celiac disease: associated disorders and survival. Gut 35:1215–1218 6. Sigurgeirsson B, Agnarsson BA, Lindelof B (1994) Risk of lymphoma in patients with dermatitis herpetiformis. BMJ 308:13–15 7. Lewis HM, Reunala TL, Garioch JJ, Leonard JN, Fry JS, Collin P, Evans D, Fry L (1996) Protective effect of gluten-free diet against development of lymphoma in dermatitis herpetiformis. Br J Dermatol 135:363–367 8. Walker-Smith JA, Guandalini S, Schmitz J, Shmerling DH, Visakorpi JK (1990) Revised criteria for diagnosis of coeliac disease. Report of working group of European Society of Paediatric Gastroenterology and Nutrition. Arch Dis Child 65:909–911 9. Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, Schuppan D (1997) Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 3:797–801 10. Blackwell PJ, Hill PG, Holmes GKT (2002) Autoantibodies to human tissue transglutaminase: superior predictors of coeliac disease. Scand J Gastroenterol 37:1282–1285 11. Volta U, Molinaro M, de Franceschi L, Fratangelo D, Bianchi FB (1995) IgA anti-endomysial antibodies on human umbilical cord tissue for celiac disease screening. Save both money and monkey. Dig Dis Sci 40:1902–1905 12. Bonamico M, Tiberti C, Picarelli A, Mariani P, Rossi D, Cipolletta E, Greco M, Di Tola M, Sabbatella L, Carabba B, Magliocca FM, Strisciuglio P, Di Mario U (2001) Radioimmunoassay to detect antitranglutaminase autoantibodies is the most sensitive and specific screening method for celiac disease. AMJ Gastroenterol 96:1536–1540 13. Catassi C, Ratsch IM, Fabiani E, Rossini M, Coppa GV, Giorgi P, Bordicchia F, Candela F (1994) Coeliac disease in the year 2000: exploring the iceberg. Lancet 343:200–203 14. Gehring AG, Crawford CG, Mazenko RS, Van Houten NJ, Brewster JD (1996) Enzyme-linked immunomagnetic electrochemical detection of Salmonella Typhimurium. J Immunol Methods 195:15–25 15. Henderson AR (1993) Assessing test accuracy and its clinical consequences: a primer for receiver operating characteristic curve analysis. Ann Clin Biochem 30:521–539 16. Swets JA (1998) Measuring the accuracy of diagnostic system. Science 240:1285–1293