Electrophoretic and immunochemical characterization of allergenic proteins in buckwheat.
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.
Yoshimasu
M A
MA
Department of Food Science, University of Guelph, Ont., Canada.
Zhang
J W
JW
Hayakawa
S
S
Mine
Y
Y
D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000009:adverse effects / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002648:Child; D002675:Child, Preschool; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D019612:Fagopyrum / Q000009:adverse effects / Q000737:chemistry*; D005260:Female; D005512:Food Hypersensitivity / Q000097:blood / Q000209:etiology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007073:Immunoglobulin E / Q000097:blood; D007074:Immunoglobulin G / Q000097:blood; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000009:adverse effects / Q000235:genetics / Q000302:isolation & purification*
D000485:Allergens; D007106:Immune Sera; D007074:Immunoglobulin G; D010940:Plant Proteins; D007073:Immunoglobulin E