Relative Quantification of Allergens
Relative levels of Ara h 1, Ara h 2 and Ara h 8 in peanut extracts were
quantified using the enzyme-linked immunosorbent assay (ELISA).
Polystyrene plates (96-well) were coated overnight at 4°C with the
different protein extracts at a range of concentrations (maximum 1 µg/mL
for Ara h 1 and Ara h 2, maximum 1 mg/mL for Ara h 8). Following
blocking with 1% BSA, rabbit anti-Ara h 1, Ara h 2 or Ara h 8
polyclonal antibody (1:1000, 50 µL/well, 2h room temperature; Indoor
Biotechnologies) was used as the primary antibody and HRP-conjugated
donkey anti-rabbit IgG monoclonal antibody (1:1,000, 50 µL/well, 1h room
temperature; BioLegend) was used for detection. Following incubation
with 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate (BioLegend), optical
density (OD) values were measured at 450 nm with reference at 570 nm.