Figure 6. ADA-a2H retains the effector functions of adalimumab. (A) Binding to membrane TNF. 293F cells that expressed TNF on the cell surface were incubated with various concentrations of adalimumab or ADA-a2H for 60 min at 4°C. The binding of the antibodies to membrane TNF was determined by a saturation binding assay using Eu-labeled goat anti-human Fc; (B) Binding to FcγRIIIA. Microtiter plates were coated with various concentrations of adalimumab or ADA-a2H at 4°C overnight. Biotin labeled FcγRIIIA (2 μg/ml) was added and incubated in the absence or presence of 1 µg/ml Ang2 at RT for 1 h. Binding of FcγRIIIA-biotin to plate bound antibodies was determined by ELISA; (C) Binding to C1q. Microtiter plates were coated with various concentrations of ADA-a2H or adalimumab overnight at 4°C. The binding of human C1q in absence or presence of 1 µg/ml Ang2 to plate bound antibodies was determined by ELISA using an HRP conjugated sheep anti-human C1q antibody. Error bars represent mean ± SEM. Results shown are representatives of two independent experiments.