A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence-Quencher Probe as a Tool for Functional Antibody Screening

J Biomol Screen. 2015 Aug;20(7):869-75. doi: 10.1177/1087057115588511. Epub 2015 May 29.


For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody-drug conjugates. Here we describe a novel activatable fluorescence-quencher pair to quantify the extent of antibody internalization and degradation in the target cells. In this assay, candidate antibodies were labeled with a fluorescent dye and a quencher. Fluorescence is inhibited outside and on the surface of cells, but activated upon endocytosis and degradation of the antibody. This assay enabled the development of a process for rapid characterization of candidate antibodies potentially in a high-throughput format. By employing an activatable secondary antibody, primary antibodies in purified form or in culture supernatants can be screened for internalization and degradation. Because purification of candidate antibodies is not required, this method represents a direct functional screen to identify antibodies that internalize efficiently early in the discovery process.

Keywords: antibody labeling; antibody screening; cell-based assay; degradation; flow cytometry; fluorescent dye; internalization; quencher dye.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / metabolism
  • Antibodies, Monoclonal / pharmacology*
  • Antineoplastic Agents / metabolism
  • Antineoplastic Agents / pharmacology*
  • Cell Line, Tumor
  • Drug Screening Assays, Antitumor / methods*
  • Flow Cytometry
  • Fluorescent Antibody Technique*
  • Fluorescent Dyes*
  • High-Throughput Screening Assays
  • Humans
  • Immunoconjugates / pharmacology
  • Protein Transport
  • Proteolysis


  • Antibodies, Monoclonal
  • Antineoplastic Agents
  • Fluorescent Dyes
  • Immunoconjugates