title: A combined biochemical, biophysical and immunological approach towards the identification of celiac disease-specific wheat antigens.
abstract: Celiac disease (CD) is an inflammatory affliction of the small bowel caused by an immunological hypersensitivity to ingested wheat antigens affecting almost 1 % of the population. The gliadin fraction of wheat has been shown to contain the pathogenic antigens which react with antibodies and T cells. However, there is only limited knowledge regarding the precise nature of the wheat antigens recognized by IgA antibodies from CD patients and diagnostic tests based on the gliadin fraction have been demonstrated to give frequently false positive results. The aim of this study was the characterization of wheat antigens specifically recognized by IgA antibodies of CD patients. We developed a combined biochemical, biophysical, and immunological approach for the identification of celiac disease-specific wheat antigens. It is based on sub-fractionation of the wheat gliadin fraction using two ion exchange chromatography steps, the localization of CD-specific antigens by immunoblotting with IgA antibodies from CD patients, subsequent digestion followed by electro spray ionization-liquid chromatography/mass spectrometry (LC-ESI-MS/MS) and N-terminal sequencing by Edman degradation. Through the sub-fractionation procedure it was possible to separate CD-specific IgA-reactive wheat antigens from other wheat antigens which were also recognized by IgA antibodies of individuals without CD or by CD patients on gluten-free diet. Analysis by LC-ESI-MS/MS and N-terminal sequencing of the sub-fractions and the proteins specifically recognized by CD patients identified certain γ-gliadins with molecular mass of 37,000 and 45,000 as CD-specific wheat antigens. The CD-specific γ-gliadins with the molecular mass of 37,000 and 45,000 should be useful to study pathomechanisms of the disease and to improve the specificity of diagnostic tests for CD.
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Centre for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria.
authors.forename: Bharani
authors.initials: B
authors.lastname: Srinivasan
authors.forename: Margarete
authors.initials: M
authors.lastname: Focke-Tejkl
authors.forename: Ines
authors.initials: I
authors.lastname: Swoboda
authors.forename: Claudia
authors.initials: C
authors.lastname: Constantin
authors.forename: Irene
authors.initials: I
authors.lastname: Mittermann
authors.forename: Sandra
authors.initials: S
authors.lastname: Pahr
authors.forename: Harald
authors.initials: H
authors.lastname: Vogelsang
authors.forename: Wolf-Dietrich
authors.initials: WD
authors.lastname: Huber
authors.forename: Rudolf
authors.initials: R
authors.lastname: Valenta
mesh_terms: D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000032:analysis* / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D002446:Celiac Disease / Q000276:immunology*; D005512:Food Hypersensitivity / Q000276:immunology*; D005903:Gliadin / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007070:Immunoglobulin A / Q000276:immunology; D012639:Seeds / Q000737:chemistry / Q000276:immunology; D014908:Triticum / Q000737:chemistry / Q000276:immunology*
chemical_list: D000941:Antigens; D007070:Immunoglobulin A; D005903:Gliadin