89 ELISA strategy searching for non-allergenic buckwheat.

W. MARUYAMA-FUNATSUKI (1), K. Fujino (2), H. Yamauchi (1), Y. Ichinose (1), K. Takata (1), M. Agatsuma (1), and H. Funatsuki (1). (1) Hokkaido National Agricultural Experiment Station, Hokkaido, Japan; (2) Hokkaido University, Hokkaido, Japan.

Buckwheat seed is a main ingredient of various buckwheat dishes. Although buckwheat allergy is not very common, buckwheat flour has been considered as a highly explosive allergen, which inspired the authors to breed buckwheat strains with less amount of allergenic proteins in their seeds. For the first step to search for natural mutants for BW24KD, which is one of major allergenic proteins binding frequently to patients’ serum IgE, the cDNA for BW24KD was isolated from immature seed cDNA library of Japanese strain ‘KITAYUKI.’ The cDNA was subcloned into a pMal plasmid vector, followed by bacteriological expression of BW24KD. Then we developed an ELISA system to conveniently quantify the BW24KD content in seed protein extract. ELISA, in which antibody to the expressed BW24KD is utilized, was optimized for conditions of antibody-dilution and of detectionso that BW24KD content of seed protein extract can be estimated from the standard curve. Screening of approx. 100 strains originating from Japan, Russia, and other countries by the developed ELISA revealed considerable genotypic differences in BW24KD content among strains. However, any strains lacking for BW24KD were not found. Further trial to screen many individual plants in each strain population is in progress.