Murine MoAb with diverse affinities for human mIgM (Cμ2) differ in capacity to induce apoptosis in Ramos B cells. Cells were incubated with MoAb HB57, MoAb Mu53, MoAb P24, or isotype control MOPC-21 (MoAb concentration of 10 μg/mL; MoAb:cell ratio = 2 μg/105 cells) for 42 hours before staining with FITC-annexin V and flow cytometric analysis. Apoptotic cells are indicated as those brightly positive for FITC-annexin V (see Materials and Methods). The intrinsic Fab′ binding affinities (Ka) of MoAbs HB57, Mu53, and P24 for B-cell mIgM are 5 × 108, 2 × 107, and approximately 2 × 106 mol/L−1, respectively.30 When the gate for annexin-positivity in the above experiment was set at a FITC-annexin intensity of 25 (horizontal log scale), Ramos cells incubated with isotype control MOPC or anti-IgM MoAbs P24, Mu53, and HB57 exhibited 4%, 4%, 90%, and 22% annexin-positive cells, respectively. Results similar to those above have been obtained in 12 replicate experiments. The difference between the percentage of annexin-positive cells in HB57 versus Mu53-treated cultures in the 12 replicate experiments was highly significant (mean ± SD of 38.2 ± 12.2 and 80.0 ± 10.3, respectively; P < .0001). Furthermore, the differences in the percentage of annexin-positive cells in HB57- or Mu53-treated cultures as compared with MOPC-treated control cultures (16.1 ± 8.8) were also both highly significant (P < .001).
