Specificity and cytotoxic effect of antibodies elicited in Balb/c mice with Rp5-L and Rp15-C. (A) Fifty microliters of a 1:20 dilution of sera drawn from BALB/c mice immunized with Rp5-L (thick continuous line) and Rp15-C (dashed line) was added to rabbit IgG-treated Daudi cells (5 × 105 cells). After a 30-minute incubation on ice, cells were washed and bound antibodies detected with an appropriate dilution of FITC-labeled affinity-purified xeno-antibodies to mouse IgG (Fc portion). Immunofluorescence was measured using a FACScan cytometer. Binding of anti-CD20 mAb 1F5 was used as positive control (dotted line). The anti-BSA serum-stained profile is indicated (thin continuous line). (B) Fifty microliters of a 1:20 dilution of anti-Rp5-L serum was added to rabbit IgG-treated Daudi cells (5 × 105 cells) previously preincubated for 30 minutes on ice with different concentrations of F(ab′)2 fragments of mAb rituximab (R). The assay was continued as described for panel A. The binding of antipeptide sera to cells preincubated with 100 μg/mL of infliximab (I100) was used as control. The fluorescence profile of cells stained with FITC-labeled probe is indicated (shaded area). (C) Ten microliters of a 2-fold dilution of DTT-treated complement-inactivated anti-Rp5-L (▴), -Rp15-C (○), and -BSA (×negative control) immune sera was added to wells of a round-bottom 96-well plate (Corning Costar) containing Raji cells. After a 30-minute incubation, an appropriate dilution of rabbit complement (BAG, Germany) was added. Cell viability and lysis were calculated as described in “CDC assay.” Lysis obtained by incubation of immune sera in the absence of complement (dashed lines) and of rituximab (500 ng) in the presence (♦) or absence (⋄) of complement were included as specificity controls.
