Characterization of protein allergens in a mouse model of wheat-induced anaphylaxis

Abstract

Abstract Wheat allergies are among the major types of food allergies that are potentially life-threatening because of anaphylaxis. Wheat contains 3 different types of protein allergens: water/saline-soluble albumins/globulins, alcohol-soluble gliadins and acid-soluble glutenins. Although gliadins have been linked to anaphylaxis, whether or not other proteins cause anaphylaxis is not well understood. Here, we sought to identify saline-soluble wheat proteins (SSWP) involved in anaphylaxis using a mouse model. Balb/cJ mice were sensitized to SSWP by four intraperitoneal (IP) injections along with alum as an adjuvant. Sensitization was confirmed by testing for SSWP-specific IgE using ELISA. Anaphylaxis was measured by hypothermia shock response. After multiple booster injections with SSWP, hyper-IgE plasma was collected and pooled to create a mini-plasma bank. Using hyper-IgE plasma, an IgE-Western blotting (WB) method was optimized. The IgE-binding proteins were identified by sequencing using LC/MS/MS method. The hyper-IgE plasma had an IgE titer of ~2560. The optimized IgE-WB included 3 overnight incubations followed by signal detection using an HRPO-based method. Sequencing of the IgE binding bands in WB identified the following proteins: globulin 3A (66 kDa), globulin 3B (57 kDa), serpin (43 kDa), glyceraldehyde-3-phosphate dehydrogenase (37 kDa), chitinase (28 kDa) and globulin 1 (25 kDa). This is the first study characterizing salt-soluble protein allergens in a mouse model of wheat-induced anaphylaxis. Funding: USDA, NIFA, Hatch project MICL02486 (Accession Number: 1012322), Agricultural and Food Research Initiative Competitive Program, grant number: 2018-67017-27876.