CDC activity over time.(A) Loss of CDC activity over time. Daudi cells were incubated with CD20 mAbs (10 μg/mL) for 15 minutes at room temperature to allow binding to cells. Cells were then washed to remove unbound CD20 mAbs, resuspended in 100 μL RPMI/10% FCS, and then incubated at 37°C. At various time points aliquots were taken, NHS (20% vol/vol) was added, and the cells were incubated at 37°C for 45 minutes. Cell lysis was assessed by flow cytometry using a PI exclusion assay, and the level of CDC expressed as PI-positive cells as percentage of total cells. Symbols: rituximab, ▪; 7D8, ▴; 2F2, ▾ ; 11B8, •, and KLH (control), ○. The results show mean ± SEM for 3 separate experiments. (B) CDC by CD20 mAbs following cross-linking. DOHH cells were incubated with CD20 mAbs (10 μg/mL) for 15 minutes at room temperature to allow binding. After washing away unbound antibody, various concentrations of sheep antihuman-κ F(ab′)2 fragments (20, 10, 5, and 0 μg/mL) were added as cross-linker, followed by the addition of normal human serum (NHS, 20% vol/vol) as a source of complement. Cell lysis was assessed by flow cytometry using a PI exclusion assay. The level of CDC is expressed as PI-positive cells as percentage of total cells. Groups: rituximab, ▪; 7D8, ▴; 2F2, ▾ ; 11B8, •; and KLH (control), ○. The figure shows the mean (± SEM) of 3 separate experiments.
