Assessment of Lupin Allergenicity in the Cholera Toxin Model: Induction of IgE Response Depends on the Intrinsic Properties of the Conglutins and Matrix Effects

Abstract

Background: The well-established murine model of IgE-mediated food allergy, based on oral administration of antigen and cholera toxin (CT), has within the previous years been used to evaluate various food proteins. Nonetheless, little knowledge on the factors that determine the allergenicity of food proteins is available so far. The use of proteins from the legume seed Lupinus albus as food ingredients calls for an evaluation of their allergenic potential, and therefore, we applied the cited model to investigate the putative allergenicity of three lupin protein preparations representing different matrices in which the four types of conglutins are present in varying concentrations. Methods: Weekly, BALB/c A mice were orally immunized with the three lupin protein products together with CT. Total specific antibodies and IgE were determined by ELISA and Western blotting. Results: A dose-dependent Ig response against the analyzed proteins was observed for all three lupin products, while IgE responses against conglutins β, γ and δ, but not against conglutin α, were primarily detected after oral administration of lupin flakes. Whereas no differences among the samples for total specific Ig responses were seen, orally administered lupin flake extracts were much more efficient in inducing a conglutin-specific IgE response compared with fractionated lupin protein products. Conclusions: Although the lupin-specific Ig response induced by coadministration of CT and lupin proteins appears to be dose dependent, the IgE response appears to depend merely on some intrinsic properties of the proteins as well as some factors of the protein matrix.

Characterization of IgE Binding to Lupin, Peanut and Almond with Sera from Lupin-Allergic Patients

“…This finding is in contrast to a previous study, which described that heat treatment of lupin seeds resulted in minimal changes in IgE recognition, except when prolonged autoclaving was applied [23]. The use of treated seeds, in contrast to the lupin-containing foods used in the current study, may explain the differences in results, because the presence of a matrix might affect the processing and thereby the allergenicity of lupin proteins [14]. In addition, in the present study IgE binding to processed lupin proteins differed among the lupin-allergic patients, implying that specific lupin allergens are differently affected by heat treatment.…”

“…The 2S albumin, δ-conglutin, which is claimed to be a potent allergen in other seeds, did not react with any of the patients’ sera in the immunoblot analyses, which is in accordance with other studies with fractionated lupin proteins [14, 15]. Unlike the results from the immunoblotting, δ-conglutin was recognised by 5 of the 6 sera in the ELISA.…”

“…In addition to the strong IgE binding towards α-conglutin, IgE binding to β-conglutin was observed with 5 of the 6 sera. To our knowledge, this finding has not previously been reported for human sera, although similar results were reported in a recently described mouse model for lupin [14]. γ-Conglutin, a minor conglutin in lupin, was poorly recognized by the patients’ IgE in the current study, as only 1 of the sera had high IgE binding to γ-conglutin.…”

“…Unlike the results from the immunoblotting, δ-conglutin was recognised by 5 of the 6 sera in the ELISA. In the lupin mouse model [14], similar IgE binding to δ-conglutin was demonstrated by ELISA, but not by immunoblot. This difference could be explained by the denaturing conditions in SDS-based electrophoresis, which may destroy IgE-binding epitopes in δ-conglutin.…”

“…Overall, the subunit structure of these conglutins appears to be complex and differences in the subunits in different Lupinus cultivars and species have been described [11, 12]. Although previous studies have been performed in which the allergenicity of lupin proteins was investigated [3, 9, 14, 15], no lupin allergens have yet been identified.…”