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.