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.