Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing

J Biol Chem. 2015 Oct 30;290(44):26457-70. doi: 10.1074/jbc.M115.676635. Epub 2015 Aug 20.


Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day.

Keywords: Bordetella pertussis; TROP2; antibody; antibody engineering; conformational epitope mapping; epitope mapping; protein-protein interaction; tumor necrosis factor (TNF).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antibodies / chemistry*
  • Antigens, Neoplasm / chemistry*
  • Antigens, Neoplasm / genetics
  • Cell Adhesion Molecules / chemistry*
  • Cell Adhesion Molecules / genetics
  • Epitope Mapping / methods*
  • Epitopes / chemistry*
  • Epitopes / genetics
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Mutagenesis
  • Tumor Necrosis Factor-alpha / chemistry*
  • Tumor Necrosis Factor-alpha / genetics


  • Antibodies
  • Antigens, Neoplasm
  • Cell Adhesion Molecules
  • Epitopes
  • TACSTD2 protein, human
  • TNF protein, human
  • Tumor Necrosis Factor-alpha