Denatured β casein

Microtiter plates (Nunc Maxisorp 96-well ELISA plates) were coated with 100 μl of denatured β-casein (Sigma-Aldrich, 10 μg/well) for hybridoma screening, or with 100 μl of the indicated concentration of native or denatured β-casein (Sigma-Aldrich) or food protein extract in coating buffer (0.1 M Na₂HPO₄ buffer pH 9.5) for 18 h at 4°C. Following incubation in blocking buffer [2% bovine serum albumin (BSA) in TBS] for 1 h at 37°C, the plates were further incubated with 0.1 μg/ml of the indicated purified mAb for 1 h at RT in blocking buffer. Following four washing steps in TBS Tween-20 0.05%, plates were further incubated for 1 h at RT with HRP goat anti-mouse IgG secondary antibody (Sigma-Aldrich) at a 1:6000 dilution in blocking buffer. Finally, plates were washed four times in TBS Tween-20 0.05% and after incubation with the substrate [0.3% H₂O₂, 0.1% 3,3',5,5'-tetramethylbenzidine (TMB) in 0.1 M citric acid pH 5] for 5 to 20 min at RT, the reaction was stopped with 0.2 M H₂SO₄. The absorbance at 450 nm was measured with a FilterMax F5 Multi-Mode microplate reader (Molecular Devices).