Figure 6.

Immunocomplex formation by the monoclonal AIGAs. (A) SEC profiles of samples (40 μl) containing a single antibody (3 μΜ) with or without IFN-γ (3 μΜ). Blue traces, mAb alone; red lines, samples with both mAb and IFN-γ; AU, absorbance unit. The assays were performed twice independently. (B) The dose dependence of immunocomplex formation was investigated with 1:0, 1:0.5, 1:1, and 1:2 ratios of site II mAb (2B6 and 2C10; 3 μM) to IFN-γ (0, 1.5, 3 or 6 μM) in SEC-UHPLC analysis. The assays were performed twice independently. (C) In vitro Fab-based neutralization in HeLa GAS reporter cells. Monovalent Fab fragments were generated by digesting the IgGs with the Pierce Fab micropreparation kit, according to the manufacturer’s instructions. HeLa GAS reporter cells (2 × 10⁴ cells) were stimulated with 118 pM (4 ng/ml) IFN-γ in the presence of serial threefold dilutions of Fab, beginning at 120 nM. The full-length antibody was used as a control, under serial threefold dilutions, beginning at 6.7 nM. The results are shown as the mean and SD for two independent experiments. The binding sites (I, II, and III) on IFN-γ are indicated next to the mAbs and Fabs.