title: A homogeneous fluorescence-based method to measure antibody internalization in tumor cells.
abstract: 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.
authors.affiliation: LI-COR Biosciences, Lincoln, NE 68504, USA. Electronic address: herbert.gong@licor.com.
authors.forename: Haibiao
authors.initials: H
authors.lastname: Gong
authors.affiliation: LI-COR Biosciences, Lincoln, NE 68504, USA.
authors.forename: Teresa
authors.initials: T
authors.lastname: Urlacher
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis / Q000378:metabolism*; D001557:Benzenesulfonates / Q000737:chemistry; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007211:Indoles / Q000737:chemistry; D000077544:Panitumumab
chemical_list: D000911:Antibodies, Monoclonal; D001557:Benzenesulfonates; D005456:Fluorescent Dyes; C562366:IRDye 800CW; D007211:Indoles; D000077544:Panitumumab; D066246:ErbB Receptors
keywords: Antibody; Fluorescence; Homogeneous assay; Internalization