Unit

UNIT 9.9 Epitope Mapping Using Gram-Positive Surface Display

  1. Johan Rockberg,
  2. John Löfblom,
  3. Barbara Hjelm,
  4. Stefan Ståhl,
  5. Mathias Uhlén

Published Online: 1 AUG 2010

DOI: 10.1002/0471142735.im0909s90

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Rockberg, J., Löfblom, J., Hjelm, B., Ståhl, S. and Uhlén, M. 2010. Epitope Mapping Using Gram-Positive Surface Display. Current Protocols in Immunology. 90:9.9:9.9.1–9.9.17.

Author Information

  1. School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden

Publication History

  1. Published Online: 1 AUG 2010
  2. Published Print: AUG 2010

Abstract

Antibodies have proven to be invaluable tools for a vast number of applications during the last decades, including protein purification and characterization, medical diagnosis and imaging, and treatment using therapeutic antibodies. No matter what the aims of the application are, the antibody's binding characteristics will still be the main features determining the assay's reliability. Here, we describe a protocol for determination of antibody-binding epitopes using an antigen-focused, library-based approach where library members are generated by fragmentation of antigen DNA and presented as cloned peptides on the cell surface of the Gram-positive bacterium Staphylococcus carnosus. The rigid cell structure of this organism allows for multivalent expression and permits rapid library analysis and sorting of antibody-binding cells using flow-sorting devices. Epitopes are determined by DNA sequencing of the sorted cells and alignment back to the antigen sequence. The protocol described here has been shown useful for mapping of both monoclonal and polyclonal binders with varying epitope lengths. Curr. Protoc. Immunol. 90:9.9.1-9.9.17. © 2010 by John Wiley & Sons, Inc.

Keywords:

  • epitope;
  • cell surface;
  • Staphylococcus carnosus;
  • epitope mapping;
  • Gram-positive;
  • antibody