A homogeneous fluorescence-based method to measure antibody internalization in tumor cells.

We have developed a simple fluorescence-based method to monitor antibody internalization. Panitumumab was dual-labeled with the fluorophore IRDye 800CW and quencher IRDye QC-1 to yield the biomolecular probe Pan800QC. The fluorescence of IRDye 800CW is quenched by IRDye QC-1 on the same intact antibody. After incubation with epidermal growth factor receptor (EGFR)-expressing cells, internalization of Pan800QC was detected by an increase in fluorescence signal due to enzymatic digestion of the antibody and separation of IRDye 800CW and IRDye QC-1. By optimizing reaction conditions, a signal-to-background ratio of 8.5 was obtained. This homogeneous assay can be applied in the characterization and screening of internalizing antibodies.