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Epitope Mapping

Identiflcation of Antibody- Binding Sites on Protein Antigens

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Part of the book series: Springer Protocols Handbooks ((SPH))

Abstract

An epitope can be simply defined as that part of an antigen involved in its recogmtion by an antibody In the case of protein antigens, an epitope would consist of a group of mdlvidual ammo-acid side-chains close together on the protein surface Epitope mapping, then, becomes the process of locating the epitope, or identifying the mdividual ammo-acids mvolved Apart from its mtrmsic value for understanding protein structure-function relationships, it also has a practical value in generating antibody probes of defined specificity as research tools and in helping to define the immune response to pathogenic proteins and organisms Some authors have even extended the epitope concept to the mteraction between peptide hormones and then receptors (1), not every immunologist would be happy about this, but it does make the point that in mapping epitopes, we are studying a biological process of fundamental importance, that of protein-protein mteraction Epitope mapping is usually done with monoclonal antibodies (MAbs), though it can be done with polyclonal antisera in a rather less rigorous way, bearing in mind that antisera behave as mixtures of MAbs. Mapping can be done directly by X-ray crystallography of antibody-antigen complexes, but it can also be done by changing individual ammo-acids, by using antigen fragments and synthetic peptides or by competition methods in which two or more antibodies compete for the same, or adjacent, epitopes. The term “epitope mapping” has also been used to describe the attempt to determine all the major sites on a protein surface that can elicit an antibody response, at the end of which one might claim to have produced an “epitope map” of the protein mununogen (2) This mformatron might be very useful, for example, to someone wishing to produce antiviral vaccines. However, there is a limit to how far one can go down this road, because the map obtained may be influenced by how MAbs are selected and by the mapping method used Furthermore, the more strictly correct definmon of epltope mapping is based on antigenicity (the ability to recognize a specific antibody).

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References

  1. Wells, J A (1995) Structural and functional epltopes in the growth hormone receptor complex Brotechnology 13,647–651

    Article  CAS  Google Scholar 

  2. Atassl, M Z (1984) Antigenic structure of proteins Eur J Biochem 145, 1–20

    Article  Google Scholar 

  3. Berzoksky, J A (1985) Intrinsic and extrmsic factors in protein antigenic structure Science 219,932–940

    Article  Google Scholar 

  4. Reece, J C., Geysen, H M, and Rodda, S J. (1993) Mapping the major human T helper epltopes of tetanus toxm The emerging picture J Immunol 151,6175–6184

    PubMed  CAS  Google Scholar 

  5. Clark, E A and Ledbetter, J A (1994) How B and T cells talk to each other Nature 367, 425–428

    Article  PubMed  CAS  Google Scholar 

  6. Howard, J C (1993) Restrlctlons on the use ofantlgemcpeptides by the nnmune system Proc Natl Acad Sci USA 90, 3777–3779

    Article  PubMed  CAS  Google Scholar 

  7. Stern, L J and Wiley, D C (1994) Antigenic peptide binding by class I and class II histocompatlbihty proteins Structure 2,245–251

    Article  PubMed  CAS  Google Scholar 

  8. Rammensee, H-G (1995) Chemistry of peptldes associated with MHC class I and class II molecules Curr Opin lmmunol 7, 85–96.

    Article  CAS  Google Scholar 

  9. van Regenmortel, M H V (1989) Structural and functional approaches to the study of protein antlgemclty Immunol Today 10, 266–272

    Article  PubMed  Google Scholar 

  10. Barlow, D J, Edwards, M S, and Thornton, J M (1986) Continuous and discontinuous protein antigenic determinants Nature 322, 747–748

    Article  PubMed  CAS  Google Scholar 

  11. Laver, W G, An-, G M, Webster, R G, and Smith-Gill, S. J (1990) Epltopes on protein antigens misconceptions and reahties Cell 61, 553–556

    Article  PubMed  CAS  Google Scholar 

  12. Amit P, Mariuzza, R, Phllips, S, and Poljak, R. (1986) Three-dlmenslonal structure of an antigen-antibody complex Science 233, 747–753

    Article  PubMed  CAS  Google Scholar 

  13. Zvl, A, Kustanovlch, I, Felgelson, D, Levy, R, Elsenstem, M, Matsushita, S, Richalet-Secordel, P, van Regenmortel, M H V, and Anghster, J (1995) NMR mapping of the antlgenic determmant recogmsed by an anti-gp120, human immunodeficlency virus neutrahzing antibody Eur J Blochem 229, 178–187

    Google Scholar 

  14. Dore, I, Weiss, E, Altschuh, D, and van Regenmortel, M H V (1988) Visuahzatlon by electron microscopy of the location of tobacco mosaic virus epltopes reacting with monoclonal antibodies Virology 162, 279–289

    Article  PubMed  CAS  Google Scholar 

  15. Tzartos, S J, Rand, D E, Emarson, B L, and Lmdstrom, J M (1981) Mapping of surface structures of Electrophorus acetylchoinie receptor using monoclonal antibodies J Biol Chem 256,8635–8645

    PubMed  CAS  Google Scholar 

  16. Le Thlet Thanh, Nguyen thl Man, Buu Mat, Phan Ngoc Tran, Nguyen thl Vinh Ha, and Morris, G. E (1991) Structural relatlonshlps between hepatltls B surface antigen in human plasma and dimers of recombmant vaccme a monoclonal antibody study Virus Res 21, 141–154

    Article  Google Scholar 

  17. MohnarO, G A and Eby, W C (1984) One antigen may form two precipltm lmes and two spurs when tested with two monoclonal antibodies by gel diffusion assays Mol Immunol 21,181–184

    Article  Google Scholar 

  18. Johne, B, Gadnell, M, and Hansen, K (1993) Epltope mappmg and bmdmg kinetlcs of monoclonal antibodies studied by real time blospeclfic interaction analysis using surface plasmon resonance J Immunol Methods 160, 191–198

    Article  PubMed  CAS  Google Scholar 

  19. Alexander, H., Alexander, S, Getzoff, E D, Tamer, J A, Geysen, H M, and Lemer, R A (1992) Altering the antlgemclty of proteins Proc Natl Acad Sci USA 89, 3352–3356

    Article  PubMed  CAS  Google Scholar 

  20. Ikeda, M, Hamano, K, and Shlbata, T (1992) Epltope mapping of anti-recA protem IgGs by region specified polymerase chain reaction mutagenesis J Blol Chem 267, 6291–4296

    CAS  Google Scholar 

  21. Wang, L F, Hertzog, P J, Galams, M, Overall, M L, Wame, G J, and Lmnane, A W (1994) Structure-functton analysis of human IFN-alpha-mapping of a conformattonal epttope by homologue scannmg J Immunol 152, 705–715

    PubMed  CAS  Google Scholar 

  22. Pmg, L H and Lemon, S M (1992) Antigemc structure of human hepatttis-A virus defined by analysts of escape mutants selected agamst murme monoclonal antibodies J Virol 66, 2208–2216

    Google Scholar 

  23. Nguyen thi Man, Cartwrtght, A J, Osborne, M, and Morris, G E (1991) Structural changes in the C-terminal region of human brain creatme kmase studted with monoclonal antibodies Biochim Biophys Acta 1076, 245–251

    Article  Google Scholar 

  24. Burnens, A, Demotz, S, Corradm, G, Binz, H, and Bosshard, H R (1987) Epitope mapping by differential chemical moditicatton of free and antibody-bound antigen Science 235,780–783

    Article  PubMed  CAS  Google Scholar 

  25. Jemmerson, R and Paterson, Y (1986) Mapping antigemc sites on a protem anttgen by the proteolysts of anttgen-antibody complexes Science 232, 1001–1004

    Article  PubMed  CAS  Google Scholar 

  26. Zhao, Y and Chatt, B. T (1995) Protein epitope mapping by mass spectrometry Anal Chem 66,3723–3726

    Article  Google Scholar 

  27. Mazzoni, M R, Malmski, J A, and Hamm, H E (1991) Structural analysts of rod GTP-binding protem, Gt Limtted proteolytic dtgestton pattern of Gt with four proteases defines monoclonal anttbody epitope J Biol Chem 266,14,072–14,081

    PubMed  CAS  Google Scholar 

  28. Ellgaard, L, Holtet, T L, Moestrup, S K, Etzerodt, M, and Thogersen, H C (1995) Nested sets of protem fragments and then use in epitope mapping-charactertzation of the epitope for the S4D5 monoclonal anttbody bmdmg to receptor-associated protem J Immunol Meth 180,53–61

    Article  CAS  Google Scholar 

  29. Morris, G E (1989) Monoclonal antibody studies of creatme kmase The ART epttope evidence for an intermediate in protein foldmg Biochem J 257, 461–469

    PubMed  CAS  Google Scholar 

  30. Morris, G E and Nguyen thi Man (1992) Changes at the N-termmus of human brain creatme kinase during a transition between inactive folding intermediate and active enzyme Biochim Bzophys Acta 1120,233–238

    Article  CAS  Google Scholar 

  31. Stanley, K K (1988) Epttope mapping using pEX Meth Mol Biol 4,351–361

    Google Scholar 

  32. Nguyen thi Man and Morris, G E (1993) Use of epttope hbrartes to identify exon-specttic monoclonal anttbodies for charactertzatton of altered dystrophms in muscular dystrophy Amer J Hum Genet 52, 1057–1066

    PubMed  CAS  Google Scholar 

  33. Wang, L F, Du Plessis, D H, White, J R, Hyatt, A R, and Eaton, B T (1995) Use of a gene-targeted phage display random epttope library to map an antrgemc determinant on the bluetongue vnus outer capsid protein VP5 J Immunol Methods 178, 1–12

    Article  PubMed  CAS  Google Scholar 

  34. Lenstra, J A, Kusters, J G,and vander Zeijst, B A M (1990) Mapping of viral epttopes with procaryotic expression systems (review) Arch Virol 110,1–24

    Article  PubMed  CAS  Google Scholar 

  35. Thanh, L T, Nguyen tht Man, Hart, S, Sewry, C A, Dubowitz V, and Moms, G E (1995) Characterization of genettc delettons in Becker Muscular Dystrophy using monoclonal antibodies against a deletion-prone region of dystrophm Amer J Med Genet 58, 177–186

    Article  Google Scholar 

  36. Burch, H B, Nagy, E V, Kam, K C, Lanar, D E, Carr, F E, Wartofsky, L, and Burman, K D(1993) Expression polymerase chain reaction for the in vttro synthesis and epttope mapping of autoanttgen Appbcatton to the human thyrotropm receptor f Immunol Methods 158,123–130

    Article  CAS  Google Scholar 

  37. Sedgwtck, S G, Nguyen tht Man, Ellis, J M., Crowne, H, and Moms, G E (1991) Rapid mapping by transposon mutagenesis of epttopes on the muscular dystrophy protein, dystrophm Nucietc Acids Res 19,5889–5894

    Article  Google Scholar 

  38. Frtguet, B, Fedorov, A N, and DJavadi-Ohamance, L (1993) In vitro gene expression for the localtzatton of antigemc determmants-apphcation to the E Coli tryptophan synthase beta2 subumt. J Immunol Methods 158, 243–249

    Article  Google Scholar 

  39. Gross, C H and Rohrmann, G F (1990) Mapping unprocessed epitopes using deletion mutagenesis of gene fusions Biotechniques 8, 196–202

    Article  PubMed  CAS  Google Scholar 

  40. Geysen, H M, Meleon, R H, and Barteling, S J (1984) Use of peptlde synthesis to probe viral antigens for epttopes to a resolution of a single amino-acid Proc Nutl Acad Sci USA 81, 3998–4002

    Article  CAS  Google Scholar 

  41. Frank, R, Kless, M, Lahmann, H, Behn, C H, and Gausepohl, H (1995) Combmatorlal synthesis on membrane supports by the SPOT techmque, in Peptides 1994 (Mala, H L S, ed), ESCOM, Leaden, pp 479–480

    Google Scholar 

  42. Holmes, C P, Adams, C L, Kochersperger, L M, Mortensen, R B, and Aldwin, LA (1995) The use of hght-directed combmatonal peptlde synthesis in epltope mapping Biopolymers 37, 199–211

    Article  PubMed  CAS  Google Scholar 

  43. Houghten, R A, Pmllla, C, Blondelle, S E, Appel, J R, Dooley, C T, and Cuervo, J H (1991) Generatlon and use of synthetic peptlde combmatorlal libraries for basic research and drug discovery Nature 354, 84–86

    Article  PubMed  CAS  Google Scholar 

  44. Scott, J K and Smith, G P (1990) Searching for peptlde hgands with an epltope library Science 249, 386–390

    Article  PubMed  CAS  Google Scholar 

  45. Morris, G E, Nguyen, C, and Nguyen thl Man (1995) Speclficlty and V, sequence of two monoclonal antibodies against the N-termmus of dystrophin Biochem J 78,355–359

    Google Scholar 

  46. Mattheakls, J C, Bhatt, R R, and Dower, W J (1994) An in vitro display system for ldentlfymg hgands from very large peptlde hbrarles Proc Natl Acad Sci USA 91, 9022–9026

    Article  Google Scholar 

  47. Lu, Z., Murray, K S, van Cleave, V, LaVallle, E R, Stahl, M L., and McCoy, J M (1995) Expression of thloredoxin random peptlde hbrarles on the Escherlchla co11 cell surface as functional fusions to flagellin Bio/technology 13, 366–372

    Article  PubMed  CAS  Google Scholar 

  48. Moms, G E (ed.) (1996), Methods in Molecular Bzology, vol 66 Epztope Mapping Protocols Humana, Totowa, NJ

    Google Scholar 

  49. Wang, K S and Strauss, J H (1991) Use of a lambda gtl 1 expression library to locahze a neutrahzing antibody-bmdmg site in glycoprotem-E2 of Smdbls virus J Virol 65,7037–7040

    PubMed  CAS  Google Scholar 

  50. Ho, D D, Fung, M S C, Cao, Y Z, LI, X L., Sun, C, Chang, T W, and Sun, N C (1991) Another dlscontmuous epltope on glycoprotem gp120 that IS important in Human Immunodeficlency Virus Type-l neutrahzatlon is identified by a monoclonal antlbody Proc Natl Acad Sci USA 88, 8949–8952

    Article  PubMed  CAS  Google Scholar 

  51. Rowlands, D J (1992) How can peptlde vaccines work FEMS Microbiol Lett 100, 479–481

    Google Scholar 

  52. Albam, S and Roudler, J (1992) Molecular basis for the assoclatlon between hladr4 and rheumatoid arthritis-from the shared epltope hypothesis to a peptldlc model of rheumatold arihrltls Clin Biochem 25,209–212

    Article  Google Scholar 

  53. Butler, M H, Sohmena, M, Dirkx, R, Hayday, A, and Decamllh, P (I993) Identificatlon of a dominant epltope of glutamic acid decarboxylase (GAD-65) recognized by autoantlbodles in Stiff-Man syndrome J Exp Med 178,2097–2106

    Article  PubMed  CAS  Google Scholar 

  54. Carson, D A (1994) The value of epltope mappmg in autoimmune diseases J Clin Invest 94, 1713

    Article  PubMed  CAS  Google Scholar 

  55. Frank, M B, Itoh, K, and McCubbm, V (1994) Epltope mappmg of the 52-kD Ro/SSA autoantigen Clin Exp Immunol 95,390–396

    Article  PubMed  CAS  Google Scholar 

  56. Palace, J, Vincent, A, Beeson, D, and Newsom-Davis, J (1994) Immunogenicity of human recombinant acetylcholme receptor alpha subumt cytoplasmlc epltopes dommate the antibody response in four mouse strams Autoimmunity 18, 113–119

    Article  PubMed  CAS  Google Scholar 

  57. Lm, M S, Ma, Y H, Hayden, M R, and Brunzell, J D (1992) Mapping of the epltope on lrpoprotem llpase recogmzed by a monoclonal antibody (5D2) which mhlblts hpase actlvlty Biochim Biophys Acta 1128, 113–115

    Google Scholar 

  58. Ptetu, G, Rtbba, A S, Cherel, G, and Meyer, D (1992) Epttope mappmg by cDNA expression of a monoclonal antibody which mhtbits the bmdmg of von Wtllebrand factor to platelet glycoprotem-IIblIIIa Biochem J 284,711–715

    Google Scholar 

  59. Landis, R C, Bennett, R I, and Hogg, N (1993) A novel LFA-1 acttvation epttope maps to the I-domain J Cell Btol 120, 1519–1527

    Article  CAS  Google Scholar 

  60. Mans, C A, Underwood, P A, Bean, P A, Sheehan, M, and Charlesworth, J A (1994) Relative topography of btologtcally active domains of human vttronectm-evidence from monoclonal antibody epttope and denaturatton studtes J Blol Chem 269, 23,845–23,852

    Google Scholar 

  61. Melhus, H, Bavik, C O, Rask, L, Peterson, P A, and Ertksson, U (1995) Epttope mapping of a monoclonal antibody that blocks the bmdmg of retmol-binding protem to its receptor Brochem Bzophys Res Commun 210, 105–112

    Article  CAS  Google Scholar 

  62. Morns, G E, Frost, L C, Newport, P A, and Hudson, N (1987) Monoclonal antibody studies of creatme kmase Antibody-binding sites in the N-terminal regton of creatme kmase and effects of anttbody on enzyme refolding Blochem J 248, 53–59

    Google Scholar 

  63. Syu, W J and Kahan, L (1992) Both ends of Eschertchra Cob rtbosomal protein-S 13 are tmmunochemically accesstble in sttu J Protein Chem 11,225–230

    Article  PubMed  CAS  Google Scholar 

  64. Nmg, G, Maunsbach, A B, Lee, Y J, and Moller, J V (1993) Topology of Na,K-ATPase alpha subumt epltopes analyzed with oltgopeptide-specific antibodies and double-labelmg nnmunoelectron mtcroscopy FEBS Lett 336,521–524

    Article  Google Scholar 

  65. Morris, G E and Cartwright, A J (1990) Monoclonal antibody studies suggest a catalytic site at the interface between domams in creatme kmase Blochim Bzophys Acta 1039,318–322

    Article  CAS  Google Scholar 

  66. Kruger, M, Wright, J, and Wang, K (1991) Nebulm as a length regulator of thm filaments of vertebrate skeletal muscles-correlatton of thm filament length, nebulm size, and epitopeprofile J Cell Biol 115,97–107

    Article  PubMed  CAS  Google Scholar 

  67. Morris, G E, Snnmons, C, and Nguyen tht Man (1995) Apo-dystrophms (Dp 140 and Dp71) and dystrophm sphcmg isoforms in developmg brain. Biochem Btophys Res Commun 215, 361–367

    Article  CAS  Google Scholar 

  68. Carter, J M (1994) Epttope predictton methods, in Peptrde Analyszs Protocols (Dunn, B M and Pennmgton, M W, eds), Humana, Totowa, NJ, pp 193–206

    Chapter  Google Scholar 

  69. Mandal, C, Kmgery, B D, Anchm, J M, Subramantam, S, and Lmthtcum, D S (1996) ABGEN. a knowledge-based automated approach for anttbody structure modellmg Nature Blotechnol 14,323–328.

    Article  CAS  Google Scholar 

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Morris, G.E. (1998). Epitope Mapping. In: Rapley, R., Walker, J.M. (eds) Molecular Biomethods Handbook. Springer Protocols Handbooks. Humana Press. https://doi.org/10.1007/978-1-59259-642-3_47

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  • DOI: https://doi.org/10.1007/978-1-59259-642-3_47

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-501-0

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