Toward the Assessment of Food Toxicity for Celiac Patients: Characterization of Monoclonal Antibodies to a Main Immunogenic Gluten Peptide Bele´ n Moro´ n1,2, Michael T. Bethune3, Isabel Comino 1 , Hamid Manyani1, Marina Ferragud 4 , Manuel Carlos Lo´ pez5, A´ ngel Cebolla4, Chaitan Khosla2,3,6, Carolina Sousa1* 1 Departamento de Microbiolog´ıa y Parasitolog´ıa, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain, 2 Department of Chemistry, Stanford University, Stanford, California, United States of America, 3 Department of Biochemistry, Stanford University, Stanford, California, United States of America, 4 Biomedal S.L., Sevilla, Spain, 5 Departamento de Biolog´ıa Molecular, Instituto de Parasitolog´ıa y Biomedicina ‘‘L o´pez-Neyra’’, Consejo Superior de Investigaciones Cient´ıficas (CSIC), Granada, Spain, 6 Department of Chemical Engineering, Stanford University, Stanford, California, United States of America Abstract Background and Aims: Celiac disease is a permanent intolerance to gluten prolamins from wheat, barley, rye and, in some patients, oats. Partially digested gluten peptides produced in the digestive tract cause inflammation of the small intestine. High throughput, immune-based assays using monoclonal antibodies specific for these immunotoxic peptides would facilitate their detection in food and enable monitoring of their enzymatic detoxification. Two monoclonal antibodies, G12 and A1, were developed against a highly immunotoxic 33-mer peptide. The potential of each antibody for quantifying food toxicity for celiac patients was studied. Methods: Epitope preferences of G12 and A1 antibodies were determined by ELISA with gluten-derived peptide variants of recombinant, synthetic or enzymatic origin. Results: The recognition sequences of G12 and A1 antibodies were hexameric and heptameric epitopes, respectively. Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1. This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes. Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody. Peptide recognition of A1 included gliadin peptides involved in the both the adaptive and innate immunological response in celiac disease. Conclusions: The sensitivity and epitope preferences of the A1 antibody resulted to be useful to detect gluten relevant peptides to infer the potential toxicity of food for celiac patients as well as to monitor peptide modifications by transglutaminase 2 or glutenases. Citation: Moro´ n B, Bethune MT, Comino I, Manyani H, Ferragud M, et al. (2008) Toward the Assessment of Food Toxicity for Celiac Patients: Characterization of Monoclonal Antibodies to a Main Immunogenic Gluten Peptide. PLoS ONE 3(5): e2294. doi:10.1371/journal.pone.0002294 Editor: Jacques Zimmer, Centre de Recherche Public-Sante´, Luxembourg Received February 22, 2008; Accepted April 13, 2008; Published May 28, 2008 Copyright: 2008 Moro´ n et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Asociacio´ n de Celiacos de Madrid (to Carolina Sousa), by the CTA (Corporacio´ n Tecnolo´ gica de Andaluc´ıa) and IDEA (Agencia de Innovaci o´n y Desarrollo de Andaluc´ıa) (to Angel Cebolla) and by grants BFU2007-64999 from Plan Nacional de Investigacio´ n cient´ıfica, Desarrollo e Innovacio´ n tecnolo´ gica (Miniterio de Educacio´ n y Ciencia) and RICET-RD06/0021-0014, Spain (to Manuel C. Lo´ pez). Bele´n Moro´ n was supported by a fellowship from Consejo Andaluz de Colegios Oficiales de Farmace´uticos. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: A´ngel Cebolla is a founder and stockholder in Biomedal S.L., which is developing the moAbs described in this article to detect gluten in food. Marina Ferragud is a staff scientist in Biomedal S.L. The rest of the authors don’t have a personal or financial conflict of interest. * E-mail: csoumar@us.es Introduction Celiac disease (CD) is a common autoimmune disorder that has genetic, environmental, and immunological components. Though under-diagnosed, it is one of the most prevalent chronic gastrointestinal diseases in humans, and exhibits unusually large clinical, histological, immunological, and genetic heterogeneity [1,2]. The clinical spectrum of CD has been expanded in recent years, with the identification of asymptomatic patients, patients with minimal symptoms (the most difficult to detect), and patients with extra-intestinal symptoms [2–5]. Regardless of symptomatic presentation, active disease in virtually all CD patients relies on dietary exposure to a common environmental antigen, gluten. The ingestion of gluten proteins contained in wheat, barley, and rye, and, in some cases, oats [6,7], leads to characteristic inflammation, villous atrophy, and crypt hyperplasia in the CD patient upper small intestine [2]. In wheat gluten, the principal toxic components belong to a family of closely related proline and glutamine rich proteins called gliadins [8]. Several epitopes responsible for the toxicity of gliadins have been identified based on their ability to stimulate proliferation of gluten-responsive DQ2 (or DQ8) restricted PLoS ONE | www.plosone.org 1 May 2008 | Volume 3 | Issue 5 | e2294