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Identification of an IgE-binding epitope of a major buckwheat allergen, BWp16, by SPOTs assay and mimotope screening.
- Source :
-
International archives of allergy and immunology [Int Arch Allergy Immunol] 2010; Vol. 153 (2), pp. 133-40. Date of Electronic Publication: 2010 Apr 21. - Publication Year :
- 2010
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Abstract
- Background: The buckwheat 16-kDa protein (BWp16), as reported in our previous study, is a major allergen in buckwheat; however, the IgE-binding epitopes of BWp16 have not as yet been identified.<br />Methods: We screened candidates for IgE-binding epitopes on BWp16 by using arrays of overlapping peptides synthesized on activated cellulose membranes (SPOTs membrane). The mimotope method was also used to analyze IgE-binding epitopes of BWp16. Nine single alanine (Ala) mutants of BWp16 expressed in Escherichia coli were used to confirm the epitopes of BWp16. The IgE-binding activity of single Ala mutants of BWp16 was determined by ELISA with mouse anti-BWp16 polyclonal antiserum or ELISA inhibition with sera from buckwheat allergic patients.<br />Results: The SPOTs assay identified amino acid residues 99-110, i.e. EGVRDLKELPSK, as a candidate for the linear IgE-binding epitope of BWp16. The mimotope method indicated that peptides similar to EGVRDLKE were candidate sequences for epitopes of BWp16. Ala scanning of rBWp16 revealed that all EGVRDLKE peptides containing a single amino acid mutation had weaker IgE-binding activity than rBWp16 WT. An ELISA inhibition assay for rBWp16 WT revealed the inhibitory effect of rBWp16 D103A to be less than that of rBWp16 WT.<br />Conclusions: We identified the peptide EGVRDLKE as a very likely candidate for the IgE-binding epitope of BWp16, and Asp103 as the critical amino acid in BWp16. This is the first report on the identification of IgE-binding epitopes of BWp16. Our findings will contribute to the production of BWp16 hypoallergens, and to allergen-specific immunotherapy for buckwheat allergy.<br /> (Copyright © 2010 S. Karger AG, Basel.)
Details
- Language :
- English
- ISSN :
- 1423-0097
- Volume :
- 153
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- International archives of allergy and immunology
- Publication Type :
- Academic Journal
- Accession number :
- 20407269
- Full Text :
- https://doi.org/10.1159/000312630