Electrophoretic and Immunochemical Characterization of Allergenic Proteins in Buckwheat

Abstract

Background: Buckwheat allergies are not common, however, it is considered to be a very potent allergen. Ingestion of small amounts has been found to produce anaphylactic reactions, particularly in children. Identification and characterization of the major allergen(s) in buckwheat are currently underway, however, there are some discrepancies in the findings. Methods: Identification of the major allergen(s) was determined through Western blotting using buckwheat-allergic patients’ sera. Once the allergenic proteins were identified, they were purified, their IgE-binding activity assessed through an indirect ELISA and the N-terminal amino acid sequence completed. To assess the stability of the IgE-binding epitopes, protein fractions were exposed to various treatments and assayed using an indirect ELISA. Lastly, the presence of anti-buckwheat IgG in the patients’ sera was analyzed through Western blotting and ELISA. Results: IgE binding was detected to proteins with molecular masses of approximately 14 and 18 kDa. N-terminal sequencing was completed and found to share some homology with rice proteins associated with rice allergies and cross-allergenicity with buckwheat proteins. When the water-soluble protein fraction was heated, exposed to acidic and alkaline conditions and fully denatured, IgE-binding activity was reduced. When the fraction was partially denatured through urea, IgE-binding activity increased. Furthermore, IgG-binding activity was detected with proteins only above the 20 kDa region. Conclusions: Proteins with molecular masses around 14 and 18 kDa were identified as the major allergenic proteins in the buckwheat-allergic patients’ sera tested in this study. Results also indicate that these two proteins possess IgE-binding capability.

Immunological Characterization and Mutational Analysis of the Recombinant Protein BWp16, a Major Allergen in Buckwheat

“…Sera from Patients with Positive IgE Binding to Buckwheat Since buckwheat contains several different allergens, [5][6][7] we performed a Western blot analysis with buckwheat extracts to identify the allergen recognized by patients' sera #001 to #015. All sera except #003 and #004 reacted to the 15-kDa band corresponding to BWp16 (Fig.…”

“…Several lines of evidence have suggested that buckwheat contains a number of allergens to react with IgE from buckwheat allergy patients. [5][6][7] One of them, the 24-kDa protein Fag e 1, has been reported to be the major allergen responsible for buckwheat allergy based on the results of IgE binding assays with sera from patients with positive IgE binding activity to buckwheat. 5,6,8) On the other hand, Tanaka et al 9) showed that the 16-kDa protein BWp16 is resistant to pepsin digestion and appeared to be responsible for IHR caused by buckwheat, whereas the 24-kDa protein was pepsin-sensitive and reacted with sera that had a positive buckwheat-specific IgE CAP RAST score, but was not responsible for IHR.…”

Buckwheat Allergy: An Emerging Clinical Problem in Europe

“…The most represented proteins are the so called "storage proteins" and in particular 13S globulins [30]. Several buckwheat proteins are described as being able to bind IgE in allergic patients, and proteins with molecular weight of 9, 16, 19, and 24 kDa are considered major allergens [31][32][33][34][35] (Table 1). It has recently been suggested that sensitization to specific buckwheat allergens would be related to specific symptoms, leading to three different clinical patterns.…”

Epitope mapping and identification on a 3D model built for the tartary buckwheat allergic protein TBb

“…In the past decades, scientists have reported the allergic composition in CB [11]. Some allergic proteins in CB with different molecular weights of 6, 9, 19, 22, 24, and 60-70 kDa have been identified [12,13].…”