Characterization of rituximab mutants. (A) Binding of 125I-labeled F(ab′)2 fragments of rituximab mutants to Daudi cells. 125I-labeled F(ab′)2 fragments of mAbs were incubated with Daudi cells for 2 hours at 37°C. The cell-bound and free 125I-labeled mAbs were then separated by centrifugation through phthalate oils and the cell pellets together with bound antibody counted for radioactivity. Data from saturation-binding experiments were analyzed by nonlinear least-squares regression for curve-fitting and dissociation constant estimation. (B) Dissociation of FITC–anti-CD20 mAbs from Raji cells. Cells were incubated with FITC-labeled anti-CD20 mAbs (10 μg/mL) at 37°C for 1 hour, washed twice, and resuspended. Samples of cells were taken at 0, 1, 2, and 4 hours and then washed and analyzed by FCM. Data are mean ± SD of at least 3 experiments.
