Determination of Specific IgE Responses
The specific IgE-binding capacity of the proteins of the various processing conditions was analyzed using the ELISA protocol described above. The serum of 4 patients highly allergic to peanuts diluted 1:1000 in 1% BSA were each used as the primary antibody (50 μL/well, 2h room temperature). Biotinylated polyclonal goat anti-human IgE antibody (1:20,000, 50 µL/well, 1h room temperature; Bethyl Laboratories Inc., TX, USA) followed by incubation with HRP-streptavidin (1:3,000, 50 µL/well, 1h room temperature; BioLegend) were used for detection.
A serial dilution of recombinant human IgE antibody at 50 ng/ml (ELISA Ready-SET-Go! Kit, Thermo Fisher Scientific, ON, Canada) binding to goat anti-human IgE capture antibody (1:1,000; Bethyl Laboratories Inc.) coated to the plate was used to construct a standard curve by plotting known concentrations versus OD values at 450 nm with reference at 570 nm.