Hrp goat anti mouse igg fcγ fragment specific secondary antibody

Microtiter plates (Nunc Maxisorp 96-well ELISA plates) were coated with 100 μl of denatured E.coli-expressed ZNS1 (500 ng/well) for hybridoma screening, or with 100 μl of the indicated concentration of denatured E.coli-expressed NS1 proteins, or native or denatured HEK293-expressed ZNS1 in coating buffer (0.1 M Na₂HPO₄ buffer pH 9.5) for 18 h at 4°C. Following incubation in blocking buffer (5% skimmed milk in TBS) for 1 h at 37°C, the plates were further incubated with hybridoma supernatants for screening, or with 0.5 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 Fcγ fragment specific secondary antibody (Jackson Immunoresearch) at a 1:6000 dilution. 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’-tetramethylbemzidine (TMB) in 0.1 M citric acid pH 5] for 10 to 30 minutes 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).

Microtiter plates (Nunc Maxisorp 96-well ELISA plates) were coated for 18 h at 4°C with 100 μl of HPV18 E6-GST (150 ng/well) for hybridoma screening, or with 100 μl of the indicated concentration of recombinant HPV E6 proteins in coating buffer (0.1 M Na₂HPO₄ buffer pH 9.5) for cross-reactivity determinations. Following incubation in blocking buffer (5% skimmed milk in TBS) for 1 h at 37°C, the plates were further incubated for 1 h at room temperature (RT) with hybridoma supernatants for screening, or with 0.5 μg/ml of 7D2 or 9E2 mAbs in blocking buffer for cross-reactivity assessment. Following four washing steps in TBS containing 0.05% Tween-20, plates were further incubated for 1 h at RT with HRP goat anti-mouse IgG Fcγ fragment specific secondary antibody (Jackson Immunoresearch) at a 1:10000 dilution. Finally, plates were washed four times in TBS with 0.05% Tween-20 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 10 minutes at RT, the reaction was stopped with 0.2 M H₂SO₄. The absorbance at 450 nm was measured with a 800TS microplate reader (BioTek).