Comparison of various concentrations of high-affinity MoAb HB57 and intermediate-affinity MoAb Mu53 for induction of apoptosis and binding to membrane IgM. (A) Apoptosis assay. Ramos B cells were cultured (22 to 24 hours) with the indicated concentrations of MoAb or medium alone before staining with FITC-annexin, as in Fig 1. The percentage of annexin-positive cells in medium cultures was subtracted from the percentage of annexin-positive cells in anti-IgM cultures to give ▵ percentage annexin-positive cells. The data shown represent the mean ± SD from two experiments. (B) Binding assay. Cells were exposed to the indicated concentrations of MoAb HB57, Mu53, or isotype control MOPC-21 for 5 minutes at 37°C, under conditions that prevent ligand internalization and capping (Materials and Methods). MoAb binding was detected by indirect immunofluorescence. The data are expressed as the MFI above the background noted with isotype control MOPC-21 (ie, ▵ MFI). Similar results to the binding experiment shown were obtained in an additional experiment performed at 37°C and in two experiments performed at 4°C. For both the apoptosis and the binding experiments, MoAb concentrations of 1, 10, and 100 μg/mL correspond to MoAb:cell ratios of 0.2 μg/105 cells, 2 μg/105 cells, and 20 μg/105 cells, respectively.
