Abstract
The residues contributing to the formation of the epitope recognized by a monoclonal antibody can be defined in several ways. Mutagenesis on the target antigen, followed by screening of the reactivity of the new variants with the antibody, is particularly powerful to reveal the functional contribution of each amino acid in the context of the native antigen. The current protocol provides a relatively simple procedure to study the surface of the target antigen in the search for residues involved in recognition. If the antigen is successfully displayed on the surface of filamentous bacteriophages, it can be quickly scanned by simultaneous mutagenesis of multiple solvent-exposed residues and high-throughput screening of the new variants with the antibody to be mapped. Once a few amino acids critically involved in recognition are defined, they can be used as starting points for a comprehensive exploration of the antigenic region by randomization of their whole neighborhood. The analysis of binding and sequence data allows delineating a detailed picture of the epitope recognized by the antibody under investigation.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Boyman O, Kovar M, Rubinstein MP et al (2006) Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science 311:1924–1927
Rojas G, Pupo A, Leon K et al (2013) Deciphering the molecular bases of the biological effects of antibodies against Interleukin-2: a versatile platform for fine epitope mapping. Immunobiology 218:105–113
Davies DR, Cohen GH (1996) Interactions of protein antigens with antibodies. Proc Natl Acad Sci U S A 93:7–12
Benjamin DC, Perdue SS (1996) Site-directed mutagenesis in epitope mapping. Methods 9:508–515
Chao G, Cochran JR, Wittrup KD (2004) Fine epitope mapping of anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display. J Mol Biol 342:539–550
Smith GP, Petrenko VA (1997) Phage display. Chem Rev 97:391–410
Böttger V, Böttger A (2009) Epitope mapping using phage display peptide libraries. Methods Mol Biol 524:181–201
Deroo S, Muller CP (2001) Antigenic and immunogenic phage displayed mimotopes as substitute antigens: applications and limitations. Comb Chem High Throughput Screen 4:75–110
Saphire EO, Montero M, Menendez A et al (2007) Structure of a high affinity “mimotope” peptide bound to HIV-1 neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies. J Mol Biol 369:696–709
Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82:488–492
Fellouse FA, Sidhu SS (2007) Making antibodies in bacteria. In: Howard GC, Kaser MR (eds) Making and using antibodies. A practical handbook. CRC Press, Boca Raton, FL, pp 157–177
Acknowledgement
Prof. Sachdev Sidhu contributed to the design of this mapping approach with helpful advice.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Rojas, G. (2014). Fine Epitope Mapping Based on Phage Display and Extensive Mutagenesis of the Target Antigen. In: Ossipow, V., Fischer, N. (eds) Monoclonal Antibodies. Methods in Molecular Biology, vol 1131. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-992-5_27
Download citation
DOI: https://doi.org/10.1007/978-1-62703-992-5_27
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-991-8
Online ISBN: 978-1-62703-992-5
eBook Packages: Springer Protocols