Eia plate

The anti-E2 antibodies were assayed for their comparative affinity for the J6CF E2-Fc and TH E2-Fc proteins. All steps were performed at room temperature. A sample of each of the various monoclonal antibodies was subjected to either a 3- or 10-fold serial dilution, and the dilutions were distributed to an EIA plate (Thermo Fisher Scientific) in which the J6CF E2-Fc or TH E2-Fc protein had been immobilized (0.5 μg/well). An antibody, HR1-007 raised against Protobothrops flavoviridis venom hemorrhagic factor was employed as a control human IgG. After incubation for 1 hour, the plates were washed with PBS containing 0.05% Tween 20 (PBS-T), and horseradish peroxidase (HRP) -conjugated goat anti-human IgG F(ab’)2 (1:5000; Thermo Fisher Scientific) was added to each well. After incubation for 1 hour, the plates were washed with PBS-T, and color was developed with ELISA POD substrate TMB solution (Nacalai Tesque, Kyoto, Japan); reactions were quenched with 1 mol/L sulfuric acid, and absorbance (at 450 nm) was measured using a microplate reader.

Antibodies were subjected to 3-fold serial dilution, starting from a concentration of 20 μg/mL. Equivalent amounts of biotinylated e2d066 IgG were added to each dilution; the mixtures then were stirred and transferred to an EIA plate (Thermo Fisher Scientific) in which the TH E2-Fc protein had been immobilized (0.5 μg/well) at room temperature (all microplate incubations were performed at room temperature unless otherwise indicated). After 1 hour, the plate was washed with PBS-T, and avidin-HRP (1:3000; GE Healthcare) was added. After 1 hour, the plate was washed with PBS-T, and color development, quenching, and plate reading were performed as described above for EIA.