Complement deposition by CD20 mAbs. (A-B) To detect C1q and C4c binding to CD20 mAbs coated on the cell surface, DOHH cells were incubated with CD20 mAbs at 10 μg/mL for 15 minutes at room temperature, followed by the addition of NHS (1% vol/vol) as a source of complement and incubation at 37°C for 10 minutes. Deposition of complement components was assessed by flow cytometry after incubation of the cells with FITC-labeled antibody against C1q (A) or C4c (B). (C-D) To assess C1q binding to CD20 mAbs coated onto ELISA plates, plates were coated with CD20 or control IgG1 mAb. Human C1q (2 μg/mL) was added to IgG-coated wells and detected with a rabbit antihuman C1q antibody followed by a peroxidase-conjugated anti-rabbit IgG-Fc antibody (D). To confirm that similar levels of CD20 mAbs were bound to the plates, they were then washed, and bound IgG was detected with an alkaline-phosphatase-conjugated goat anti-human IgG (C). Symbols: KLH (control), ○; rituximab, ▪; 7D8, ▾ ; 2F2, ▴; and 11B8, •. Data show OD values in the ELISA systems and represent 1 of 3 separate experiments yielding similar results.