Ability of cross-linking to enhance Triton X-100 insolubility and CDC activity of different antigens.
(A) To assess whether more antigen could be moved into the Triton X-100–insoluble fraction by additional cross-linking, EHRB cells were incubated with FITC-mAbs as before, washed, and then divided into 4. Two of these samples were incubated with goat anti–mouse IgG F(ab′)2 fragments for 15 minutes on ice to facilitate cross-linking. After washing, one of the cross-linked and one of the non–cross-linked samples were lysed in Triton X-100 and washed as detailed above, and the remaining 2 samples retained to determine total binding with and without cross-linking. Results were then expressed as the percent of antigen retained in the Triton X-100–insoluble fraction using the equations: (MFI Triton X-100 insoluble/MFI total) × 100 and (MFI cross-linked Triton X-100/MFI cross-linked total) × 100 for non–cross-linked and cross-linked, respectively. Error bars represent SD of results for at least 3 experiments. (B) To assess whether cross-linking could enhance CDC activity, mAb (10 μg/mL) was bound to cells for 15 minutes at room temperature as before and then washed once, prior to addition of cross-linking agent at a range of concentrations (0, 5, 1, 0.2 μg/mL) for 10 minutes at room temperature. NHS (5% vol/vol) was then added and CDC measured as described in the legend to Figure 1. Similar data were obtained in 3 experiments.
