Monoclonal Antibodies to Recombinant Fag e 3 Buckwheat Allergen and Development of a Two-site ELISA for Its Quantification

PURPOSE Buckwheat is a major cause of anaphylaxis, and Fag e 3 is the key major allergen in buckwheat. However, an immunoassay system for the quantification of Fag e 3 has yet to be developed. METHODS We developed a 2-site enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (mAbs) produced against recombinant Fag e 3. We applied this ELISA to quantify native Fag e 3 in total buckwheat extract. RESULTS Four clones of mAbs were produced, and all recognized vicilin allergens not only from buckwheat, but also from peanut and walnut. However, the ELISA using these antibodies was only able to quantify Fag e 3 in the total extract after addition of 1% sodium dodecyl sulphate (SDS) and heating, which facilitated dissociation of the allergen. The detection limit of the developed 2-site ELISA was 0.8 μg/mL. The measurement of Fag e 3 in the total extract of buckwheat showed that approximately 12% of protein in total buckwheat extract was Fag e 3. CONCLUSIONS We have developed an ELISA system for the quantification of the group 3 buckwheat allergen, Fag e 3, specifically. This assay will be useful for standardization of buckwheat allergens and monitoring of buckwheat contamination in foods.