Identification of IgE-binding proteins in salmon and grass carp
The extracted salmon and grass carp proteins were analyzed on SDS-PAGE (Figure 1A). Sera from 45 subjects (30 samples from HK and 15 samples from Japan; marked with asterisks* in Supplementary Tables E1 & E2) and non-atopic controls (n = 2) were used to investigate IgE-binding to salmon and grass carp proteins by western blotting (Figure 1B & C). No IgE binding was observed for control sera. Serum samples from Japanese fish allergic subjects showed more diverse IgE binding compared to those from HK, including to grass carp extract despite of the lower IgE level measured on ImmunoCAP. For both populations, the 11kDa band in salmon and 10kDa band in grass carp represented the major IgE-binding protein. In HK fish allergic subjects, 18/30 (60.0%) and 16/30 (53.3%) showed IgE binding to this 10kDa protein in grass carp and salmon respectively, while 10/15 (66.7%) subjects in Japan had a positive reaction to the 10kDa proteins in salmon and grass carp extracts. Six (40.0%) Japanese subjects reacted to proteins with molecular sizes 130 kDa and 58 kDa.
Protein bands that showed IgE reactivity to at least three serum samples were then excised from SDS-PAGE for allergen identification on mass spectrometry. These included the 130kDa, 43kDa, 34kDa, 25kDa and 11kDa bands from salmon extract, as well as the 43kDa, 34kDa and 10kDa bands from grass carp extract. For salmon extract, the 130kDa protein was identified as α-collagen, 58kDa band as β-enolase, 32kDa band as aldolase A, 25kDa band as GAPDH and the 11kDa band as β-parvalbumin (Supplementary Table E3). Similarly, the 58kDa, 36kDa and 10kDa bands from grass carp extract corresponded to β-enolase, GAPDH and β-parvalbumin, respectively.