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Epitope Mapping: B-cell Advanced article Epitopes Article Contents . Introduction GE Morris, Wolfson Centre for Inherited Neuromuscular Disease RJAH Orthopaedic Hospital, . What Is a B-cell Epitope? . Epitope Mapping Methods Oswestry, UK and Keele University, Keele, Staffordshire, UK . Applications Immunoglobulin molecules are folded to present a surface structure complementary to doi: 10.1002/9780470015902.a0002624.pub2 a surface feature on the antigen – the epitope is this feature of the antigen. Epitope mapping is the process of locating the antibody-binding site on the antigen, although the term is also applied more broadly to receptor–ligand interactions unrelated to the immune system. Introduction formed of highly convoluted peptide chains, so that resi- dues that lie close together on the protein surface are often Immunoglobulin molecules are folded in a way that as- far apart in the amino acid sequence (Barlow et al., 1986). sembles sequences from the variable regions of both the Consequently, most epitopes on native, globular proteins heavy and light chains into a surface feature (comprised of are conformation-dependent and they disappear if the up to six complementarity-determining regions (CDRs)) protein is denatured or fragmented. Sometimes, by acci- that is complementary in shape to a surface structure on the dent or design, antibodies are produced against linear antigen. These two surface features, the ‘paratope’ on the (sequential) epitopes that survive denaturation, though antibody and the ‘epitope’ on the antigen, may have a cer- such antibodies usually fail to recognize the native protein. tain amount of flexibility to allow an ‘induced fit’ between The simplest way to find out whether an epitope is confor- them. In the narrow sense, epitope mapping is the process mational is by Western blotting after SDS-PAGE. If the of locating the antibody-binding site on the antigen. The antibody still binds after the protein has been denatured, term is also applied in a broader sense to T-cell epitopes the epitope is unlikely to be highly conformational. It must (Stern and Wiley, 1994) and has even been applied to be remembered, however, that few proteins are completely receptor–ligand interactions unrelated to the immune sys- linearized by denaturation and some epitopes identified by tem. Only B-cell epitopes are relevant to the biochemical Western blotting may still have a conformational element. understanding of antibody–antigen interactions. See also: See also: Proteins: Fundamental Chemical Properties; Antibodies; Immunoglobulin Fold: Structures of Proteins Protein Folding In Vivo; Western Blotting in the Immunoglobulin Superfamily Epitope Mapping Methods WhatIsaB-cellEpitope? X-ray crystallography is a ‘structural’ approach to epitope mapping that can identify contact residues in antigen–an- In the case of protein antigens, B-cell epitopes consist of tibody complexes, but needs good crystals of complexes groups of amino acids that lie close together on the protein and has usually been used for highly conformational surface and that determine antigenicity. Epitope mapping epitopes on the surface of soluble proteins. Nuclear mag- is usually done with monoclonal antibodies (mAbs), netic resonance (NMR) is another ‘structural’ approach though it can be done with polyclonal antisera in a rather that is performed in solution and thus avoids the need for less specific way. See also: Antigens; Epitopes; Monoclonal crystals. Since NMR is limited to peptides and small pro- Antibodies teins, it may be unsuitable for highly assembled epitopes. Van Regenmortel 1989 made the important distinction Electron microscopy is a low-resolution method of ‘struc- between ‘structural’ epitopes that are ‘in contact’ with the tural mapping’ which, although unable to identify contact antibody, as defined by X-ray crystallography and ‘func- residues, can be used to identify the location of epitopes on tional’ epitopes defined by amino acid residues that are very large antigens such as whole viruses. See also: Electron important for binding the antibody and cannot be Microscopy; Macromolecular Structure Determination by replaced. X-ray Crystallography; Nuclear Magnetic Resonance It is also essential to distinguish between conformational (NMR) Spectroscopy of Proteins (‘discontinuous’, ‘assembled’) epitopes, in which amino The remaining epitope mapping methods are essentially acids far apart in the protein sequence are brought together ‘functional’ in approach and can be divided into four by protein folding, and linear (‘continuous’, ‘sequential’) groups: (1) competition methods, (2) antigen modification epitopes, which can often be mimicked by simple peptide methods, (3) fragmentation methods and (4) the use of sequences (Berzoksky, 1985). Most native proteins are synthetic peptides or peptide libraries. ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net 1 Epitope Mapping: B-cell Epitopes Competition methods can be very useful when a rela- nucleotide sequence (Wilson et al.,2001).See also:Phage tively low degree of mapping resolution is adequate. You Display Technologies may want to establish, for example, that two mAbs recog- Overlapping peptides for mapping sequential epitopes nize different, nonoverlapping epitopes for a two-site can be synthesized on pins (PEPSCAN), on a cellulose immunoassay, or to find mAbs against several different membrane support (SPOTS) or on micro-arrays. Combi- epitopes on the same antigen so that results due to cross- natorial libraries of random peptide sequences in solution reactions with other proteins can be rigorously excluded. can also be used. The advent of random peptide libraries The principle behind competition methods is to determine displayed on the surface of filamentous bacteriophage took whether two different mAbs can bind to a monovalent an- this approach a vital step further by enabling selection of tigen at the same time (in which case they must recognize displayed peptides instead of screening. Selection of ran- different epitopes) or whether they compete with each other dom peptides is unique in producing a range of sequences for the same antigen epitope. See also: Immunoassay that are related, but not identical, to the antigen sequence; Protection of the antigen from acetylation by the pres- this enables inferences to be made about which amino acids ence of antibody is applicable to conformational epitopes in the epitope are most important for antibody binding. and is considered more reliable than direct chemical mod- Peptide libraries can also be displayed on the surface of ification, which may affect antigen structure and give false Escherichia coli in the major flagellum component, flag- positive results. Protection from proteolytic digestion, also ellin. An advantage shared by all peptide methods is that known as ‘protein footprinting’, is similar in principle; an- antigen is not required and this may be important for ‘rare’ tigens are exposed to proteases in the presence or absence of antigens that are difficult to purify. Phage-displayed pep- antibody (which is fairly protease-resistant) and differences tides may also mimic conformational epitopes although in digestion are detected by gel electrophoresis. If the an- they do not correspond to any linear sequence in the an- tigen can be expressed from recombinant cDNA and the tigen (‘mimotopes’). Computational methods are being approximate position of the epitope is known, specific mu- developed both to identify conformational epitopes (Haste tations can be introduced by site-directed mutagenesis Andersen et al., 2006) and to predict immunogenic epitopes methods. Alternatively, random mutations can be intro- (Flower, 2003). See also: Bacteriophage Display of duced into part of the antigen by polymerase chain reaction Combinatorial Antibody Libraries (PCR), followed by screening to detect epitope-negative There is increasing interest in developing mass spectro- mutants. The ‘escape mutant’ approach for viral surface metric (MS) approaches to epitope mapping because of di- epitopes recognized by neutralizing antibodies involves se- rect sequencing capability, applicability to conformational lection and sequencing of spontaneous mutants whose in- and posttranslationally modified epitopes and the possibil- fectivity is no longer blocked by the antibody. ities for automation (Hager-Braun and Tomer, 2005). In A simple fragmentation approach for epitopes that sur- general, tryptic peptides that bind to antibody, or which are vive denaturation is partial protease digestion of the an- protected by antibody from hydrogen–deuterium exchange, tigen, followed either by Western blotting for larger are identified by LC-MS-MS, but 100% coverage of antigen fragments or by high-performance liquid chromatography sequence with tryptic peptides is often hard to achieve. (HPLC). The fragments that bind antibody can be iden- tified by N-terminal microsequencing or by mass spect- rometry. Chemical cleavage at specific amino acids is a Applications commonly used alternative method for generating antigen fragments. See also: Liquid Chromatography; Mass Apart from its intrinsic value for understanding protein Spectrometry in Biology interactions, epitope mapping also has a practical value in If the antigen can be expressed from recombinant cDNA, generating antibody probes of defined specificity as re- digestion with DNAase I, followed by cloning and expres- search tools and in helping to define the immune response sion of the cDNA fragments to create ‘epitope libraries’,

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