Abstract
The unique property of specific high-affinity binding to more or less any target of interest has made antibodies tremendously useful in numerous applications. Hence knowledge of the precise binding site (epitope) of antibodies on the target protein is one of the most important features for understanding its performance and determining its reliability in immunoassays. Here, we describe a high-resolution method for mapping epitopes of antibodies based on bacterial surface expression of antigen fragments followed by antibody-based flow cytometric sorting. Epitopes are determined by DNA sequencing of the sorted antibody-binding cells followed by sequence alignment back to the antigen sequence. The method described here has been useful for the mapping of both monoclonal and polyclonal antibodies with varying sizes of epitopes.
The authors Anna-Luisa Volk and Francis Jingxin Hu have contributed equally to this work.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Geysen HM, Meloen RH, Barteling SJ (1984) Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc Natl Acad Sci U S A 81:3998–4002
Sheriff S, Silverton EW, Padlan EA et al (1987) Three-dimensional structure of an antibody-antigen complex. Proc Natl Acad Sci U S A 84:8075–8079
Herzenberg LA, Parks D, Sahaf B et al (2002) The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem 48:1819–1827
Mattanovich D, Borth N (2006) Applications of cell sorting in biotechnology. Microbial Cell Fact 5:12
Rockberg J, Löfblom J, Hjelm B et al (2008) Epitope mapping of antibodies using bacterial surface display. Nat Methods 5
Augustin J, Götz F (1990) Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation. FEMS Microbiol Lett 54:203–207
Rockberg J, Löfblom J, Hjelm B et al (2010) Epitope mapping using gram-positive surface display. Curr Protoc Immunol Chapter 9:Unit9.9. doi: 10.1002/0471142735.im0909s90
Hudson EP, Uhlen M, Rockberg J (2012) Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes. Sci Rep 2:706
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
Volk, AL., Hu, F.J., Rockberg, J. (2014). Epitope Mapping of Monoclonal and Polyclonal Antibodies Using Bacterial Cell Surface Display. 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_29
Download citation
DOI: https://doi.org/10.1007/978-1-62703-992-5_29
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