Epitopes (the binding sites on the protein) can be divided into linear and conformational. Linear epitopes are formed by a continuous sequence of amino acids in a protein, while conformational epitopes are composed of amino acids that are discontinuous in the protein sequence but are brought together upon three-dimensional protein folding. The vast majority of antigen-antibody interactions have conformational epitopes.
Methods for epitope mapping
Epitope mapping of complex target antigens, such as integral membrane proteins or multi-subunit proteins, is often challenging because of the difficulty in expressing and purifying these types of antigens.
There are several methods available for mapping antibody epitopes on target antigens :
- The gold standard approach is X-ray co-crystallography, which allows direct visualization of the interaction between the antigen and antibody. However, this approach is technically challenging, requires large amounts of purified protein, and can be time-consuming and expensive.
- Array-based oligo-peptide scanning (sometimes called overlapping peptide scan or pepscan analysis): This technique uses a library of oligo-peptide sequences from overlapping and non-overlapping segments of a target protein and tests for their ability to bind the antibody of interest. This method is fast and relatively inexpensive, and specifically suited to profile epitopes for large number of candidate antibodies against a defined target. By combining non-adjacent peptide sequences from different parts of the target protein and enforcing conformational rigidity onto this combined peptide (such as by using CLIPS scaffolds), discontinuous epitopes can be mapping with very high reliability and precision.
- Site-directed mutagenesis: Using this approach, systematic mutations of amino acids are introduced into a protein sequence followed by measurement of antibody binding in order to identify amino acids that comprise an epitope. This technique can be used to map both linear and conformational epitopes, but is labor-intensive and slow, typically limiting analysis to a small number of amino acid residues.
- Mutagenesis Mapping. This approach utilizes a comprehensive mutation library, with each clone containing a unique amino acid mutation and the entire library covering every amino acid in the target protein. Amino acids that are required for antibody binding can be identified by a loss of reactivity and mapped onto protein structures to visualize epitopes. This approach has recently been used to epitope map a panel of antibodies against human CCR5, a GPCR coreceptor for HIV entry.
- Other methods, such as phage display, and limited proteolysis, provide high throughput but lack reliability, especially for conformational epitopes.
- Gershoni, JM; Roitburd-Berman, A; Siman-Tov, DD; Tarnovitski Freund, N; Weiss, Y (2007). "Epitope mapping: The first step in developing epitope-based vaccines". BioDrugs 21 (3): 145–56. doi:10.2165/00063030-200721030-00002. PMID 17516710.
- Westwood, MR; Hay, FC (2001). Epitope Mapping: a practical approach. Oxford, Oxfordshire: Oxford University Press. ISBN 0-19-963652-4.[page needed]
- "Mapping Epitopes with H/D-Ex Mass Spec". 2011.
- Gaseitsiwe, S.; Valentini, D.; Mahdavifar, S.; Reilly, M.; Ehrnst, A.; Maeurer, M. (2009). "Peptide Microarray-Based Identification of Mycobacterium tuberculosis Epitope Binding to HLA-DRB1*0101, DRB1*1501, and DRB1*0401". Clinical and Vaccine Immunology 17 (1): 168–75. doi:10.1128/CVI.00208-09. PMC 2812096. PMID 19864486.
- Linnebacher, Michael; Lorenz, Peter; Koy, Cornelia; Jahnke, Annika; Born, Nadine; Steinbeck, Felix; Wollbold, Johannes; Latzkow, Tobias et al. (2012). "Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: A pilot study with colorectal carcinoma patient samples". Analytical and Bioanalytical Chemistry 403 (1): 227–38. doi:10.1007/s00216-012-5781-5. PMID 22349330.
- Timmerman (2009). "Functional Reconstruction of Structurally Complex Epitopes using CLIPSTM Technology#". The Open Vaccine Journal. doi:10.2174/1875035401002010056.
- Cragg, M. S. (2011). "CD20 antibodies: Doing the time warp". Blood 118 (2): 219–20. doi:10.1182/blood-2011-04-346700. PMID 21757627.
- http://www.integralmolecular.com/download/INTG-AN-Shotgun%20Mutagenesis.pdf[full citation needed]
- Banik, Soma S. R.; Doranz, Benjamin J. (2010). "Mapping Complex Antibody Epitopes". Genetic Engineering and Biotechnology News 3 (2): 25–8.
- Paes, Cheryl; Ingalls, Jada; Kampani, Karan; Sulli, Chidananda; Kakkar, Esha; Murray, Meredith; Kotelnikov, Valery; Greene, Tiffani A. et al. (2009). "Atomic-Level Mapping of Antibody Epitopes on a GPCR". Journal of the American Chemical Society 131 (20): 6952–4. doi:10.1021/ja900186n. PMC 2943208. PMID 19453194.