Figure 3.

Panitumumab and trastuzumab are specific for human EGFR and HER2 and can bind their targets concurrently. (A) Simultaneous binding of panitumumab and trastuzumab to SW780 cells using immunocytochemistry. Panitumumab and trastuzumab were conjugated to Alexa488 and Alexa594, respectively. About, 3 × 10⁴ SW780 cells and NIH/3T3 cells were then incubated for 3 hours at room temperature with 50 μg/ml and 100 μg/ml of pan-Alexa488 and tra-Alexa594, respectively. NIH/3T3 cell line was used as a negative control. NIH/3T3 showed fluorescence in Hoechst 33342 (nuclear stain) but no fluorescence signals for Pan-Alexa488 and Tra-Alexa594, confirming the target specificity of the antibodies. SW780 cells showed fluorescence in both pan-Alexa488 and tra-Alexa594, and co-staining on same cells, confirming antibody specificity and their ability to bind targets concurrently. (B) Determining pan IR700 and tra IR700 concentrations to saturate binding sites. About, 1 × 10⁶ cells/100 µL were incubated for 1 hour at room temperature with either 0 µg, 5 µg, 15 µg, 50 µg, 75 µg, or 100 µg of pan IR700 alone, tra IR700 alone, or in combination (pan IR700 + tra IR700). The cells were washed and IR700 fluorescence was read using APC-Cy-7 filter of BD FACSCanto II flow cytometer machine. Flow cytometry shows that the antibody binding sites saturate about 15 µg/100 µL for both the antibodies. It also confirms concurrent binding of both antibodies to their targets on the same cell.