Figure 3. Time course of internalization of immunotoxins. Xenograft D2224MG (A) and cells D336MG (B) were incubated with 50 nM Alexa-488-labeled DmAb14m-IT or DmAb14-IT at 37°C for different times. After anti-Alexa-488 quenched the cell surface fluorescence, the internalized immunotoxins were measured. In both cells DmAb14m-IT showed significantly higher internalization rate than that of the DmAb14-IT. The data shown represent the mean ± SD of three similar experiments. Scatchard analysis. Defined affinity determination for 125I-labeled DmAb14m-IT vs. ganglioside-positive D336MG cells in contrast with gangliosides-negative HEK293 cells. The saturation curve was presented. (C) Scatchard plots for activity of the percentage bound. (D) Binding affinity graph showing KD = 2.6 × 10−9 M.