A Simple and Sensitive High-Content Assay for the Characterization of Antiproliferative Therapeutic Antibodies

SLAS Discov. 2017 Mar;22(3):309-315. doi: 10.1177/1087057116677821. Epub 2016 Dec 13.


Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2'-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell-based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2'-deoxyuridine (EdU) and 4',6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs.

Keywords: EdU; cell-based assays; high-content screening; proliferation; therapeutic antibodies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents, Immunological / pharmacology*
  • Biomarkers, Tumor / genetics*
  • Biomarkers, Tumor / metabolism
  • Cell Cycle Checkpoints / drug effects
  • Cell Cycle Checkpoints / genetics*
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Deoxyuridine / analogs & derivatives
  • Deoxyuridine / chemistry
  • Female
  • Gene Expression
  • High-Throughput Screening Assays*
  • Humans
  • Indoles / chemistry
  • Mammary Glands, Human / drug effects
  • Mammary Glands, Human / metabolism
  • Mammary Glands, Human / pathology
  • Receptor, ErbB-2 / antagonists & inhibitors
  • Receptor, ErbB-2 / genetics*
  • Receptor, ErbB-2 / metabolism
  • Sensitivity and Specificity
  • Trastuzumab / pharmacology*


  • Antineoplastic Agents, Immunological
  • Biomarkers, Tumor
  • Indoles
  • DAPI
  • ERBB2 protein, human
  • Receptor, ErbB-2
  • 5-ethynyl-2'-deoxyuridine
  • Trastuzumab
  • Deoxyuridine