Electrochemical immunosensor for the diagnosis of celiac disease M.I. Pividori * , A. Lermo, A. Bonanni, S. Alegret, M. del Valle Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain article info Article history: Received 26 November 2008 Available online 27 February 2009 Keywords: Celiac disease Electrochemical immunosensor Immunoassay Transglutaminase IgA antibody abstract A novel electrochemical immunosensing strategy for the detection of antibodies to tissue transglutamin- ase (tTG) in human serum is presented. The proposed immunosensor consists of the immobilization by physical adsorption of tTG from guinea pig liver on graphite–epoxy composite (GEC) electrodes. After the reaction with the human serum (containing the specific antibodies in the case of celiac disease), the electrode was incubated with different kinds of secondary labeled antibodies, namely, horseradish peroxidase (HRP)-conjugated goat antibodies to human whole immunoglobulins (Igs), to human IgG, and finally to human IgA. Among the different classes of antibodies in human serum toward tTG, the best results were achieved when anti-tTG IgA antibodies were investigated. In total, 10 positive and 10 neg- ative serum samples were processed, obtaining a sensitivity of 70% and a specificity of 100% compared with the commercial enzyme-linked immunosorbent assay (ELISA) method performed in a hospital lab- oratory. This strategy offers great promise for a simple, cost-effective, and user-friendly analytical method that allows point-of-care diagnosis of celiac disease. Ó 2009 Elsevier Inc. All rights reserved. Celiac disease is an autoimmune-mediated disorder that affects primarily the gastrointestinal tract [1]. It is characterized by chronic inflammation of the small intestinal mucosa that may re- sult in atrophy of intestinal villi, deficient adsorption of nutrients, and a variety of clinical manifestations that may begin during childhood or adult life [2,3]. There is a strong genetic predisposi- tion to celiac disease, with the major risk being attributed to the specific genetic markers known as human leukocyte antigen (HLA) 1 –DQ2 and HLA–DQ8 that are present in affected individuals [4]. Dietary proteins present in wheat, barley, and rye, commonly known as glutens, interact with these HLA molecules to activate an abnormal mucosal immune response and induce tissue damage. Most affected individuals experience remission after excluding glu- ten from their diets. Population-based studies using various combinations of sero- logical testing and small intestinal biopsy suggest that the preva- lence of celiac disease is in the range of 0.5 to 1.0% in Europe and the United States. Patients with celiac disease tend to have other chronic diseases in which the immune system attacks their own cells and tissues, including type 1 diabetes, autoimmune thyroid disease, autoimmune liver disease, rheumatoid arthritis, Addison’s disease, and Sjögren’s syndrome. Autoantibody production is an important feature of all these autoimmune disorders, signifying a breakdown of immune tolerance to self-antigens. In celiac disease, autoantibody reactivity to transglutaminase 2 (TG2) has been shown to closely correlate with the acute phase of the disease [5]. It serves as a specific and sensitive marker of celiac disease and is highly useful in aiding diagnosis and follow-up. The ability of TG2 to crosslink gliadin peptides to itself and to other protein substrates supports the hypothesis that this event is responsible for the humoral autoimmune response in celiac disease [6]. Im- mune reactivity to other autoantigens, including transglutaminase 3, has also been reported in celiac disease. The recent identification of these autoantigens involved in ce- liac disease has led to the development of new serologic diagnostic tests, but the appropriate use of these new testing strategies has not been well defined. In fact, there is no available test that can definitively diagnose or exclude celiac disease in every individual. Up to now, only the combination of clinical features and laboratory tests may confirm a diagnosis of celiac disease. Moreover, these diagnostic tests need to be performed while the patient is on a glu- ten-containing diet. The first step in pursuing a diagnosis of celiac disease is a sero- logic test. Based on very high sensitivities and specificities, the best available tests are the immunoglobulin IgA anti-human tissue transglutaminase (tTG or TG2) [7] and IgA endomysial antibody immunofluorescence (EMA) [8,9] tests that appear to have equiva- lent diagnostic sensitivity and specificity (tTG is the specific pro- tein that is identified by the IgA–EMA) [10]. Anti-gliadin antibody (AGA) tests are no longer routinely recommended be- cause of their lower sensitivity and specificity [11,12]. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.02.026 * Corresponding author. Fax: +34 93 581 2379. E-mail address: isabel.pividori@uab.cat (M.I. Pividori). 1 Abbreviations used: HLA, human leukocyte antigen; TG2, transglutaminase 2; Ig, immunoglobulin; tTG (or TG2), tissue transglutaminase; EMA, endomysial antibody immunofluorescence; AGA, anti-gliadin antibody; GEC, graphite–epoxy composite; HRP, horseradish peroxidase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; RSD, relative standard deviation. Analytical Biochemistry 388 (2009) 229–234 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio