B Cell Epitope Mapping Using Synthetic Peptides

John Mark Carter1, Larry Loomis‐Price2

1 Palo Alto, California, 2 Montgomery College Biotechnology Institute, Conroe, Texas
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 9.4
DOI:  10.1002/0471142735.im0904s60
Online Posting Date:  May, 2004
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Abstract

Synthetic peptides are used for identification of functional B cell epitopes in antibody preparations. ELISA‐type assays are used to identify sequences of proteins comprising antibody‐binding regions. This unit describes three peptide display formats. Pepscan (pins) and SPOTs (cellulose membranes) may be used as solid‐phase support media for peptide synthesis, followed by ELISA directly on the resulting peptide array. Alternatively, peptides may be cleaved from the array and tested in a standard microplate‐based antibody capture ELISA format. The discussion includes choosing the peptide sequences by length and overlap, as well as determination of the minimum essential sequence for antibody binding. This method is highly effective for continuous epitopes and is often also useful for discontinuous epitopes.

Keywords: epitope mapping; synthetic peptide; pepscan; SPOT peptide; parallel synthesis; continuous epitope; ELISA; affinity purification

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: ELISA for Mapping B Cell Epitopes Using Pin‐Bound Peptides
  • Support Protocol 1: Disruption of Pin‐Bound Peptides
  • Alternate Protocol 1: ELISA for Mapping B Cell Epitopes Using Soluble Peptides
  • Support Protocol 2: Data Analysis
  • Basic Protocol 2: ELISA for Mapping B Cell Epitopes Using Peptide Spots
  • Support Protocol 3: Stripping a Peptide Spots Array
  • Support Protocol 4: Elution of Antibody Reagent from Specific SPOTs or Pins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: ELISA for Mapping B Cell Epitopes Using Pin‐Bound Peptides

  Materials
  • Blocking solution (see recipe)
  • Pin‐bound peptide array (Mimotopes), disrupted as described (see protocol 2)
  • 1 µg/ml primary antibody (Ab) or antiserum diluted 1:1000 in blocking solution
  • PBST: 0.1% (v/v) Tween 20 in PBS ( appendix 2A)
  • Alkaline phosphatase (AP)‐conjugated secondary antibody (Kirkegaard & Perry Laboratories), diluted to manufacturer's recommendation in blocking solution
  • Soluble substrate solution (see recipe)
  • 96‐well microplates, some optically clear
  • Microplate‐size containers for solution incubations and washes
  • Rocker or orbital shaker
  • Plate reader (e.g., Spectramax; Molecular Devices)

Support Protocol 1: Disruption of Pin‐Bound Peptides

  Materials
  • Pin‐bound peptide array (Mimotopes), new or previously used for ELISA (see protocol 1)
  • Sonication buffer (see recipe) in high‐power ultrasonic bath (Blackstone∼NEY Ultrasonics), 65°C
  • Methanol, reagent grade, boiling (∼61°C) in explosion‐proof bath (e.g., Electrowave)
  • Plastic storage bags (preferably zippered)

Alternate Protocol 1: ELISA for Mapping B Cell Epitopes Using Soluble Peptides

  • 5 µg/ml streptavidin solution (see recipe)
  • 20 to 50 µg/ml peptide (soluble) solutions in PBS ( appendix 2A) or other mild buffer
  • Alkaline phosphatase stop solution (see recipe)
  • Polyethylene minitubes (8 × 12 format) and multichannel pipettor or liquid handler or 1.5‐ to 3‐ml microcentrifuge tubes, with caps
  • Plastic storage bags (preferably zippered) or humidified chamber
  • Microplate washer, optional

Support Protocol 2: Data Analysis

  Materials
  • Peptide SPOT array (Jerini Peptide Technologies or Sigma‐Genosys)
  • Blocking solution (see recipe), diluted 1:10 in water
  • 1 µg/ml primary antibody (Ab) or antiserum (diluted 1:1000 in blocking solution)
  • TBST or PBST: 0.1% (v/v) Tween 20 in TBS ( appendix 2A) or PBS ( appendix 2A), respectively
  • Alkaline phosphatase (AP)‐conjugated secondary Ab (Kirkegaard & Perry Laboratories), diluted to manufacturer's recommendation in blocking solution
  • Precipitating substrate solution (see recipe)
  • Stop solution: 1% (v/v) acetic acid
  • Container with lid, large enough to fit SPOT array with at least 1 cm free on each edge
  • Rocker platform or orbital mixer
  • Array scanner (e.g., BAS, FLA, FineScan, or Quattro; Fuji)

Basic Protocol 2: ELISA for Mapping B Cell Epitopes Using Peptide Spots

  Materials
  • SPOTs array used for ELISA (see protocol 5)
  • N,N‐Dimethylformamide (DMF), reagent grade
  • Reagent A (see recipe)
  • Reagent B: 50:40:10 (v/v/v) ethanol/water/acetic acid, made fresh daily
  • Methanol, reagent grade
  • Container large enough to fit SPOTs array with at least 1 cm free on each edge
  • Rocker platform

Support Protocol 3: Stripping a Peptide Spots Array

  Materials
  • Pin‐bound peptide array or SPOTs array used for ELISA (see protocol 1Basic Protocols 1 and protocol 52)
  • TBST or PBST: 0.1% (v/v) Tween 20 in TBS ( appendix 2A) or PBS ( appendix 2A), respectively
  • Elution buffer (see recipe)
  • Neutralization buffer: 1 M Tris·Cl, pH 7.5 ( appendix 2A)
  • PBS ( appendix 2A) or other appropriate buffer for dialysis, optional
  • Stem Recycler (Mimotopes), optional
  • 1.5‐ml microcentrifuge tubes with caps
  • Paper hole punch or small scissors, for SPOTs array only
  • Vortex mixer
  • Dialysis equipment, optional
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Figures

Videos

Literature Cited

   Atassi, M.Z. 1984. Antigenic structures of proteins. Eur. J. Biochem. 145:1‐20.
   Birk, H.W. and Koepsell, H. 1987. Reaction of monoclonal antibodies with plasma membrane proteins after binding on nitrocellulose: Renaturation of antigenic sites and reduction of nonspecific antibody binding. Anal. Biochem. 164:12‐22.
   Borràs, E., Martin, R., Judkowski, V., Shukaliak, J., Zhao, Y., Rubio‐Godoy, V., Valmori, D., Wilson, D., Simon, R., Houghten, R., and Pinilla, C. 2002. Findings on T cell specificity revealed by synthetic combinatorial libraries. J. Immunol. Methods 267:79‐97.
   Carter, J.M. 1994. Epitope prediction methods. In Methods in Molecular Biology, Vol. 36: Peptide Analysis Protocols (B.M. Dunn, and, M.W. Pennington, eds.) pp. 193‐205. Humana Press, Totowa, N.J.
   Carter, J.M., James, M., Korenstein, B., Herrero, J., Miller, B., Hu, H., Zhitnitsky, R., Luczak, C., and Chen, L. 2001 A comprehensive database of protein‐protein interactions. In Peptides: The Wave of the Future, Proceedings of the 17th American Peptide Symposium (M. Lebl and R.A. Houghten, eds.) pp. 1057‐1059. Mayflower Scientific, Kingswinford, U.K.
   Frank, R. 1992. SPOT synthesis: An easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48:9217‐9232.
   Geerligs, H.J., Weijer, W.J., Bloemhoff, W., Welling, G.W., and Welling‐Wester, S. 1988. The influence of pH and ionic strength on the coating of peptides of herpes simplex virus type 1 in an enzyme‐linked immunosorbent assay. J. Immunol. Methods 106:239‐244.
   Geysen, H.M., Meloen, R.H., and Barteling, S.J. 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.
   Geysen, H.M., Rodda, S.J., and Mason, T.J. 1986. A priori delineation of a peptide which mimics a discontinuous antigenic determinant. Mol. Immunol. 23:709‐715.
   Geysen, H.M., Rodda, S.J., Mason, T.J., Tribbick, G., and Schoofs, P.G. 1987. Strategies for epitope analysis using peptide synthesis. J. Immunol. Methods 102:259‐274.
   Houghten, R.A., DeGraw, S.T., Bray, M.K., Hoffman, S.R., and Frizell, N.D. 1986. Simultaneous multiple peptide synthesis: The rapid preparation of large numbers of discrete peptides for biological, immunological, and methodological studies. BioTechniques 4:522‐528.
   Jemmerson, R. 1987. Antigenicity and native structure of globular proteins: Low frequency of peptide reactive antibodies. Proc. Natl. Acad. Sci. U.S.A. 84:9180‐9184.
   Korth, C., Stierli, B., Streit, P., Moser, M., Schaller, O., Fischer, R., Schulz‐Schaeffer, W., Kretschar, H., Raeber, A., Braun, U., Ehrensperger, F., Hornemann, S., Glockshuber, R., Riek, R., Billeter, M., Wüthrich, K., and Oesch, B. 1997. Prion (PrPSc)‐specific epitope defined by a monoclonal antibody. Nature 390:74‐77.
   Lam, K.S., Salmon, S.E., Hersh, E.M., Hruby, V.J., Kazmierski, W.M., and Knapp, R.J. 1991. A new type of synthetic peptide library for identifying ligand binding activity. Nature 354:82‐84.
   Loomis‐Price, L.D., Levi, M., Burnett, P.R., van Hamont, J.E., Shafer, R.A., Wahren, B., and Birx, D.L. 1997. Linear epitope mapping of humoral responses induced by vaccination with recombinant HIV‐1 envelope protein gp160. J. Ind. Microbiol. Biotechnol. 19:58‐65.
   Maeji, N.M., Valerio, R.M., Bray, A.M., Campbell, R.A., and Geysen, H.M. 1994. Grafted supports used with the multipin method of peptide synthesis. Reactive Polym. 22:203‐212.
   Reineke, U., Sabat, R., Misselwitz, R., Welfe, H., Volk, H.D., and Schneider‐Mergener, J. 1999. A synthetic mimic of a discontinuous binding site on interleukin‐10. Nat. Biotechnol. 17:271‐275.
   Rodda, S.J. 2002. Peptide libraries for T cell epitope screening and characterization. J. Immunol. Methods 267:71‐77.
   Tegge, W., Frank, R., Hofman, F., and Dostman, W.R.G. 1995. Determination of cyclic nucleotide‐dependent protein kinase substrate specificity by use of peptide libraries on cellulose paper. Biochemistry 34:10569‐10577.
   Tribbick, G. 2002. Multipin peptide libraries for antibody and receptor epitope screening and characterization. J. Immunol. Methods 267:27‐35.
   Tribbick, G., Triantafyllou, B., Lauricella, R., Rodda, S.J., Mason, T.J., and Geysen, H.M. 1991. Systematic fractionation of serum antibodies using multiple antigen homologous peptides as affinity ligands. J. Immunol. Methods 139:155‐66.
   Ward, C.W., Gough, K.H., Rashke, M., Wan, S.S., Tribbick, G., and Wang, J.‐X. 1996. Systematic mapping of potential binding sites for Shc and Grb2 SH2 domains on Insulin receptor substrate‐1 and the receptors for insulin, EGF, PDGF, and FGF. J. Biol. Chem. 271:5603‐5609.
   Weiner, A.J., Geysen, H.M., Christopherson, C., Hall, J.E., Mason, T.J., Saracco, G., Bonino, F., Crawford, K., Marion, C.D., Crawford, K.A., Brunetto, M., Barr, P.J., Miyamura, T., McHutchinson, J., and Houghton, M. 1992. Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: Potential role in chronic HCV infections. Proc. Natl. Acad. Sci. U.S.A. 89:3468‐3472.
Key References
   Frank, R. 2002. The SPOT‐synthesis technique: Synthetic peptide arrays on membrane supports—principles and applications. J. Immunol. Methods 267:13‐26.
  Recent review of SPOT technology.
   Geysen et al., 1987. See above.
  Seminal publication on multiple‐peptide synthesis.
   Tribbick, 2002. See above.
  Recent review of pin technology.
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