Phage Display Selection, Analysis, and Prediction of B Cell Epitopes

Natalia Tarnovitski Freund1, David Enshell‐Seijffers2, Jonathan M. Gershoni1

1 Tel Aviv University, Tel Aviv, Israel, 2 Harvard Medical School, Charlestown, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 9.8
DOI:  10.1002/0471142735.im0908s86
Online Posting Date:  August, 2009
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Abstract

Combinatorial phage display libraries of random peptides can be used to discover the epitopes of antibodies through a procedure termed “biopanning.” The affinity isolation of phage‐displayed epitope peptidomimetics allows molecular definition of the epitopes of monoclonal antibodies (MAbs). Panels of MAb‐specific peptides allow computational prediction of B cell epitopes. Epitope profiles recognized by polyclonal serum samples can also be generated. Detailed step by step protocols and discussion of applications are provided. Curr. Protoc. Immunol. 86:9.8.1‐9.8.30. © 2009 by John Wiley & Sons, Inc.

Keywords: epitope mapping; mimotope; peptide mimetics; phage display; random peptide libraries; Mapitope; Pepitope

     
 
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Table of Contents

  • Introduction
  • General Considerations
  • Basic Protocol 1: Affinity Selection of Antibody‐Specific Phages (“Biopanning”)
  • Support Protocol 1: Determination of Phage Titer
  • Support Protocol 2: Confirmation of Positive Phages
  • Alternate Protocol 1: Amplification of Positive Phages: Multiple Rounds of Biopanning
  • Basic Protocol 2: Phage Characterization and Epitope Mapping
  • Basic Protocol 3: Construction and Use of Custom‐Tailored Phages
  • Support Protocol 3: Streptavidin Purification of Inserts
  • Support Protocol 4: Phenol: Chloroform Extraction
  • Support Protocol 5: Calibration of Insert to Vector Ratio
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Affinity Selection of Antibody‐Specific Phages (“Biopanning”)

  Materials
  • Protein G (Sigma, cat. no. P‐5170), 1 mg/ml in 1× TBS (stock solution); working solution is freshly prepared, 50 µg in 700 µl TBS (0.071 µg/µl)
  • Blocking solution A (see recipe)
  • 1× Tris‐buffered saline (TBS; see recipe), sterile
  • Antibody: monoclonal antibody (MAb) or polyclonal antibody to be screened
  • Random peptide phage display library (see )
  • Elution buffer (see recipe)
  • Neutralizing buffer (see recipe)
  • 10% (w/v) sodium azide stock solution (optional)
  • LB medium (see recipe)
  • E. coli F+ strain: DH5α F′ or K91KAN (see Table 9.8.1)
  • Terrific broth (see recipe)
  • LB/tet agar plates (see recipe)
  • Tetracycline
  • Assay controls: e.g., the cognate antigen or an antibody compatible with the secondary probe
  • Positive controls (e.g., phages from previous experiments) and negative controls (e.g., fth‐1 phages without recombinant PVIII proteins)
  • PEG/NaCl solution (see recipe)
  • Blocking solution B (see recipe)
  • Secondary antibody: anti–mouse IgG HRP conjugate (e.g., from goat)
  • ECL substrate kit (GE Healthcare)
  • 35‐mm, 6‐well cluster tissue culture plates (Corning Costar, cat. no. 3516)
  • Rockers/shakers: rockers tend to be best for small volumes; orbital or reciprocal shakers are sometimes preferred as indicated
  • Bacterial shaker‐incubator
  • 50‐ml conical tube or flask
  • 96‐well U‐bottom microtiter plate, sterile (e.g., Corning Costar, cat. no. 3799)
  • Sterile wooden toothpicks
  • Humidified chamber (unit 3.17)
  • Centrifuge with 96‐well microtiter plate adapter
  • 96‐well flat‐bottom microtiter plate, sterile (e.g., Greiner, cat. no. 655180)
  • 96‐well plate shaker/vibrator: e.g., Tetramax 100 (Heidolph Instruments, http://www.heidolph.com/)
  • Nitrocellulose membrane filters (0.45 µm; e.g., Schleicher & Schuell, cat. #10 401 169)
  • Dot‐blot vacuum manifold system
  • Additional reagents and equipment for determining phage titer ( protocol 2)
    Table 9.8.1   MaterialsGenotypes of E. coli Bacterial Strains

    Strain Genotype
    K91KAN A derivative of K91 (Hfr‐Cavalli, thi) in which the “mini‐Kan hopper” element was inserted in the lacZ gene of K91, rendering this strain kanamycin resistant
    DH5 aF′ F′ endA1 hsdR17 (r K m K+)s up E44 thi‐1 recA1 gyrA(Nalr)r elA1 2(lacIZY A‐argF)U169 deoR (φ80dlac2 (lacZ)Ml5)
    MC1061 Fa raD1392(ara‐leu)7696 galE15 galK162(lac)X74 rps L(Strr)h sdR2(r km k+)mcrA mcrBl

Support Protocol 1: Determination of Phage Titer

  Materials
  • LB/Kan: LB medium (see recipe) containing 50 µg/ml kanamycin
  • E. coli K91KAN (see Table 9.8.1)
  • Terrific broth (see recipe)
  • Eluted phages (see protocol 1, step 10)
  • 1× Tris‐buffered saline (TBS; see recipe)
  • LB medium (see recipe)
  • LB/tet agar plates (see recipe)
  • Bacterial shaker‐incubator
  • 50‐ml tubes

Support Protocol 2: Confirmation of Positive Phages

  Materials
  • Master plate ( protocol 1)
  • Terrific broth (see recipe)
  • PEG/NaCl solution (see recipe)
  • 1× Tris‐buffered saline (TBS; see recipe)
  • Sterile wooden toothpicks
  • Bacterial shaker‐incubator
  • Centrifuge
  • 2‐ml microcentrifuge tubes

Alternate Protocol 1: Amplification of Positive Phages: Multiple Rounds of Biopanning

  • TYx2 medium (see recipe)
  • Phages from an earlier round of biopanning (see protocol 1, step 10)
  • Tetracycline
  • 1× Tris‐buffered saline (TBS; see recipe)
  • 500‐ and 2000‐ml Erlenmeyer flasks, sterile
  • 250‐ to 500‐ml centrifugation bottles, sterile
  • 50‐ml centrifuge tubes, sterile
  • Refrigerated centrifuge and rotor capable of accommodating 250‐ to 500‐ml centrifugation bottles (e.g., Sorvall centrifuge with GS‐3 and SS‐34 rotors)

Basic Protocol 2: Phage Characterization and Epitope Mapping

  Materials
  • Phage display vector: e.g., fth‐1
  • Restriction enzymes: SfiI and BglI
  • Oligonucleotides (biotinylated on 5′ end; two are required):
    • A long sense strand that contains the unique segment of the insert flanked between two constant sequences that include BglI sites (compatible with the SfiI sites of the vector)
    • A short antisense oligonucleotide that complements the 3′ constant region of the former oligonucleotide
  • Klenow fragment of DNA polymerase and 10× Klenow buffer
  • 10 mM dNTP mix (i.e., 10 mM each of dATP, dCTP, dGTP, and dTTP)
  • 0.5 M EDTA
  • 70% ethanol
  • 10 mM Tris⋅Cl, pH 8 ( appendix 2A)
  • T4 DNA ligase (e.g., NEB, cat. no. 202 S) and ligase buffer
  • Electrocompetent E. coli MC1061 (see Table 9.8.1)
  • SOC medium (see recipe)
  • TYx2 medium (see recipe) containing 20 µg/ml tetracycline
  • LB‐tet agar plates (see recipe)
  • LB medium (see recipe)
  • Chroma spin column: Chroma spin+TE‐1000 (Clontech, cat. no. K1324‐2)
  • 85°C heating block
  • 16°C water bath
  • Electroporation unit (see, e.g., unit 10.15)
  • Additional reagents and equipment for basic molecular biology techniques (see Chapter 10 in this manual and Ausubel et al., ), purification of inserts with streptavidin‐agarose beads ( protocol 7), phenol/chloroform extraction of DNA ( protocol 8), and calibration of insert‐to‐vector ratio ( protocol 9)

Basic Protocol 3: Construction and Use of Custom‐Tailored Phages

  Materials
  • Streptavidin‐agarose beads (e.g., Sigma, cat. no. S‐1638)
  • TE buffer ( appendix 2A) containing 0.1 M NaCl
  • Cut insert (see protocol 6, step 12)

Support Protocol 3: Streptavidin Purification of Inserts

  Materials
  • Phenol reagent (e.g., Biophenol; Biosolve B.V., cat. no. 16912344; http://biosolve‐chemicals.com/)
  • Purified insert ( protocol 7)
  • 24:1 (v/v) chloroform/isoamyl alcohol

Support Protocol 4: Phenol: Chloroform Extraction

  Materials
  • Insert ( protocol 6)
  • Cut vector ( protocol 6)
  • T4 DNA ligase and 10× ligase buffer (e.g., NEB, cat. no. 202 S)
  • E. coli DH5α F′ competent bacteria (see Table 9.8.1)
  • LB‐tet agar plates (see recipe)
  • 16°C water bath
  • Additional reagents and equipment for transformation of E. coli by heat shock (Seidman et al., )
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Figures

Videos

Literature Cited

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   Halperin, I., Wolfson, H., and Nussinov, R. 2003. SiteLight: Binding‐site prediction using phage display libraries. Protein Sci. 12:1344‐1359.
   Hoess, R., Brinkmann, U., Handel, T., and Pastan, I. 1993. Identification of a peptide which binds to the carbohydrate‐specific monoclonal antibody B3. Gene 128:43‐49.
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Key References
   Barbas et al., 2001. See above.
  This is an extremely comprehensive laboratory manual that covers the complete spectrum of phage display technologies. It also provides excellent discussion of the variety of topics of phage display applications as well as systematic recipes for reagents and protocols.
   Model and Russel, 1988. See above.
  A fundamental text covering the basic biology of filamentous bacteriophages.
   Enshell‐Seijffers et al., 2001. See above.
  A detailed description of the construction and characteristics of the fth‐1 vector.
Internet Resources
  http://www.biosci.missouri.edu/smithgp/
  Smith Lab Web site (Dr. George Smith). This is the original set of protocols and reagents that much of the field uses. The extensive and detailed descriptions of procedures, reagents and libraries are basic for all those working with phage display systems.
  http://t.caspur.it/meps/
  MEPS Web site.
  http://web.kuicr.kyoto‐u.ac.jp/∼hjian/mimox/cgi‐bin/mimosa.pl
  MIMOX Web site.
  http://pepitope.tau.ac.il/
  Pepitope Web site.
  http://www.rasmol.org/
  RasMol Web site.
  http://www.rasmol.org/software/RasMol_Latest_Manual.html
  RasMol manual.
  http://sourceforge.net/projects/rastop
  RasTop Web page.
  http://www.rcsb.org
  RCSB (Research Collaboratory for Structural Bioinformatics) PDB (Protein Data Bank).
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