Binding properties of 22-20 and 20-22. (A) Competition ELISA showing relative binding avidity of v-mab, 22-20, 20-22, and 20-20 for binding to WR2, the anti-Id antibody to v-mab. (B) Competition ELISA showing relative binding avidity of e-mab, 22-20, 20-22, and 22-22 for binding to WN, the anti-Id antibody to e-mab. (C) Cell-binding analysis by flow cytometry. Raji cells were preincubated with CH1-DDD2-Fab-v-mab, CH1-DDD2-Fab-e-mab, or both at 1 mg/mL before staining with PE-22-20, PE-20-22, PE-v-mab, or PE-e-mab. (D) Analysis of dissociation rates from live Raji. Cells were saturated with PE-mAbs, and the fluorescence intensity was measured over time by flow cytometry. Percentage maximal binding (MFI T = 0) was calculated by dividing MFI T = x into MFI T = 0 and plotted versus time. (E) Internalization of PE mAbs was measured using flow cytometry by comparison of the MFI of trypsinized cells versus control cells after a 1-hour incubation at 37°C.
