Immunoblotting and immunoblot inhibition
A 2D-PAGE analysis of wheat and barley extract showed multiple bands (Fig 3A). Serum from the seven patients excluding cases 3 and 9 was used for immunoblotting with the wheat extract, and several IgE-binding spots were found. (Fig 3B, Fig S1). In all patients, the 2D-PAGE revealed a strong IgE binding to a 30–50 kDa wheat extract protein, which was estimated to be gliadin based on the molecular weights described in the literature.14-17 In IgE-immunoblotting using barley extract, strong bands at 30–35, 40–45, 60–80 kDa were detected in all seven patients. In addition, various other spots were detected in each patient.
In immunoblot inhibition, the bands of wheat were inhibited albeit weakly by barley extracts. On the other hand, most of the spots of barley were inhibited even by a low concentration of wheat and barley extract.