C3b, C1q binding, and CDC activity of anti-CD20 mAb. (A-B) EHRB cells were incubated with various mAbs (10 μg/mL) for 15 minutes prior to addition of 20% NHS. Samples were taken after 5 minutes of incubation at 37°C and washed, and C3b and C1q binding was assessed by staining with an anti-C3b (A) or anti-C1q (B) FITC-labeled mAb for 15 minutes at 4°C. Bars represent the mean MFI ± SD for at least 3 experiments. (C-D) To assess whether cross-linking could enhance C1q and CDC activity, mAbs were bound to EHRB cells for 15 minutes at room temperature at concentrations adjusted to provide equivalent levels of Fc at the cell surface. This concentration was determined by titrating primary mAb concentrations to a level that gave equivalent surface binding as measured with the use of a secondary antimouse IgG FITC mAb and flow cytometry. Cells were then washed once, and the sample was divided into 2 samples, one half receiving cross-linking agent 25 μg/mL (x-link) and the other receiving PBS (No-x-link) for 10 minutes at room temperature. NHS (20%) was then added, and the extent of C1q bound (C) and cell lysis (D) apparent after 5 minutes at 37°C was measured. Samples were assayed for lysis by PI staining.