ORIGINAL ARTICLE EXPERIMENTAL ALLERGY AND IMMUNOLOGY The high molecular weight glutenin subunit Bx7 allergen from wheat contains repetitive IgE epitopes A. Baar 1,2 , S. Pahr 1,2 , C. Constantin 1 , S. Giavi 3 , N. G. Papadopoulos 3 , A. S. Pelkonen 4 , M. J. M€ akel€ a 4 , S. Scheiblhofer 5 , J. Thalhamer 5 , M. Weber 1 , C. Ebner 6 , A. Mari 7 , S. Vrtala 1,2 & R. Valenta 1 1 Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology & Immunology, Vienna General Hospital, Medical University of Vienna; 2 Christian Doppler Laboratory for the Development of Allergen Chips, Medical University of Vienna, Vienna, Austria; 3 Allergy and Immunology Research Centre, University of Athens, Athens, Greece; 4 Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, Finland; 5 Department of Molecular Biology, University of Salzburg, Salzburg; 6 Ambulatory for Allergy and Clinical Immunology, Vienna, Austria; 7 Associated Centers for Molecular Allergology, Rome, Italy To cite this article: Baar A, Pahr S, Constantin C, Giavi S, Papadopoulos NG, Pelkonen AS, M€ akel€ a MJ, Scheiblhofer S, Thalhamer J, Weber M, Ebner C, Mari A, Vrtala S, Valenta R. The high molecular weight glutenin subunit Bx7 allergen from wheat contains repetitive IgE epitopes. Allergy 2014; 69: 1316–1323. Keywords allergy; component-resolved diagnosis; recombinant wheat allergens; repetitive IgE epitope; wheat food allergy. Correspondence Rudolf Valenta, MD, Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology & Immunology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. Tel.: +4314040051080 Fax: +4314040051300 E-mail: rudolf.valenta@meduniwien.ac.at Accepted for publication 16 June 2014 DOI:10.1111/all.12464 Edited by: Thomas Bieber Abstract Background: Wheat is one of the most common food allergen sources for children and adults. The aim of this study was to characterize new wheat allergens using an IgE discovery approach and to investigate their IgE epitopes. Methods: A cDNA expression library representing the wheat transcriptome was constructed in phage lambda gt11 and screened with IgE antibodies from wheat food allergic patients. IgE-reactive cDNA clones coding for portions of high molecular weight (HMW) glutenin subunits were identified by sequence analysis of positive clones. IgE epitopes were characterized using recombinant fragments from the HMW Bx7 and synthetic peptides thereof for testing of allergic patients’ sera and in basophil degranulation assays. Results: We found that the major IgE-reactive areas of HMW glutenins are located in the repetitive regions of the protein and could show that two inde- pendent IgE-reactive fragments from HMW Bx7 contained repetitive IgE epi- topes. Conclusions: Our results demonstrate that IgE antibodies from wheat food allergic patients can recognize repetitive epitopes in one of the important wheat food allergens. Recombinant HMW Bx7 may be included into the panel of allergens for component-resolved diagnosis of wheat food allergy. Wheat food allergy may be induced by direct sensitization involving class I food allergens via the gastrointestinal tract (1) involving often linear IgE epitopes, whereas respiratory allergens often contain discontinuous and/or conformational IgE epitopes (2). Sequential IgE-reactive peptide epitopes have been reported for the major peanut allergens, Ara h 1 and Ara h 2 (3, 4), for cow’s milk allergens (5, 6), certain tree nut allergens (7), nonspecific lipid transfer proteins in fruits (8), and wheat allergens (9). However, for certain class I food allergens such as the major fish allergen, parvalbumin, and certain milk allergens (e.g., alpha-lactal- bumin), also conformational IgE epitopes have been described (10, 11). To identify IgE epitopes in wheat, we used a discovery approach. For this purpose, a wheat seed cDNA library containing the wheat transcriptome was constructed and screened with serum IgE from wheat food allergic patients with the aim to identify new wheat allergens (12). Seven inde- pendent IgE-reactive cDNA clones coded for fragments of high molecular weight (HMW) glutenin subunits, which typi- cally consists of a signal peptide, an N-terminal nonrepetitive region, a large repetitive region, and a C-terminal nonrepeti- tive region (13). Interestingly, each of the IgE-reactive clones was located in the repetitive regions of the HMW subunits. A detailed analysis with recombinant fragments and synthetic peptides revealed the presence of repetitive IgE epitopes in the HMW glutenin subunit Bx7. Our finding that allergens can contain repetitive IgE epitopes provides a novel mecha- nism for the cross-linking of allergic effector cells and may explain the allergenic potency of wheat. Allergy 69 (2014) 1316–1323 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1316