Measurement of antibody titers by ELISA.
IgG1 and IgG2a titers were measured by direct ELISA: 96-well plates were coated overnight at 4°C with cashew protein extract (5µg/mL, protein equivalent) or with anti-IgG1 or anti-IgG2a (5 or 1 µg/mL, respectively) for binding of standards. Plates were washed in PBS 1X-Tween® 0.05% and blocked 1 hour at room temperature (RT) with phosphate 0.1 M pH 7.4-NaCl 0.15 M-BSA 0.1%-sodium azide 0.01% (EIA buffer). Plates were then incubated overnight at 4°C with mouse plasma (in duplicates) or serially diluted standards (Rabbit F(ab’)2 anti-mouse IgG1 or Human anti-mouse IgG2a, Bio-Rad), and then 1 hour at 37°C with relevant secondary antibodies conjugated to alkaline phosphatase (AP) (Bio-Rad). Finally, plates were incubated for 30 minutes at RT with AP substrate (p-Nitrophenyl Phosphate, pNPP) and optical densities (O.D.) at 405 nm were recorded. Antibody concentrations were calculated with Boltzmann sigmoidal equation using O.D. obtained from standards (GraphPad Prism®), and after subtracting O.D. obtained from non-specific points. IgE titers were measured by indirect ELISA to avoid competition with IgG1 binding: 96-well plates were coated with 2µg/mL of rat anti-mouse IgE (Bio-Rad) and incubated overnight at 4°C. Plates were washed in PBS 1X-Tween® 0.05% and blocked 1 hour at RT with EIA buffer. Following washing, plates were incubated overnight at 4°C with mouse plasma (in duplicates) or serially diluted standard (mouse IgE, Bio-Rad), and then 1 hour at RT with biotinylated cashew proteins for plasma samples or biotin anti-mouse IgE (1:2000, Biolegend) for standard. Plates were finally incubated 1 hour at RT with AP-conjugated streptavidine (1:5000, Jackson Immuno Research Lab), and then 15 minutes at RT with pNPP. O.D. were recorded and antibody concentrations were measured as described above.