Methods: Protein extracts from boiled, roasted, and pickled walnuts were compared with that of raw walnut using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Raw walnut protein extracts were immunoblotted with individual sera from 11 children with levels of walnut specific IgE of 0.7kU/L or higher (ImmunoCAP, ThermoFisher Scientific, Waltham, Mass). Additionally, we produced peanut extract and sera were tested with enzyme linked immunosorbentassay inhibition test.
Results: At least 8 or more distinct protein bands (9-108kDa) were visible from raw walnut by SDS-PAGE. The 9-kD (Jug r 3) and 16-kDa (Jug r 1) bands were stable in boiled and roasted walnut. The intensity of9-kD band was even enhanced in roasted and boiled walnut, whereas that of 16-kDa band was slightly decreased in roasted walnut. Meanwhile, the 28-kDa and 60-kDa (Jug r 4) bands profoundly disappeared in roasted walnut. No protein bands were shown in the walnut treated by vinegar for one month.By IgE-immunoblotting with individual sera from walnut-sensitive children, at least 8 protein components were identified. The 60-kDa band corresponding to Jug r 4 wasreacted with 72.5% of patients and the 16-kDa band (Jug r 1) and 9-kDa band (Jug r 3) were reacted with 54.5% of patients. Walnut in the IgE-ELISA was not inhibited by peanut extract.
Conclusion: This study indicatesthat IgE-binding bands suspected as Jug r 1,Jug r 3, and Jug r 4 are major problematic allergens in walnut allergic children. Also, the amount of components weighted 28kDa, 47kDa, 60kDawere reduced by heating whereas that of 9kDa and 16kDa were relatively stable.