Using Phage Display in Neurobiology

Andrew Bradbury1, Daniele Sblaterro1, Roberto Marzari2, Louise Rem3, Hennie Hoogenboom3

1 International School for Advanced Studies (SISSA), Trieste, Italy, 2 Università di Trieste, Trieste, Italy, 3 Department Pathologie Academisch Ziekenhius Maastricht and TargetQuest, Maastricht, The Netherlands
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.18
DOI:  10.1002/0471142301.ns0518s18
Online Posting Date:  May, 2002
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

In this unit some of the basic protocols involved in the manipulation of phage display libraries are described, including the rescue and amplification of such libraries, selection and screening from them and testing of derived clones.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Concentration of Phage or Phagemid Particles by PEG Precipitation
  • Support Protocol 1: Preparing Helper Phage
  • Basic Protocol 2: Rescuing Phage/Phagemid Particles from Libraries
  • Basic Protocol 3: Selection of Phage Antibodies to an Antigen Immobilized Indirectly or Directly on (Plastic) Surfaces
  • Alternate Protocol 1: Selection of Phage Antibodies Using Biotinylated Antigen and Streptavidin‐Paramagnetic Beads
  • Support Protocol 2: Rescue of Phage/Phagemid by Infection of E. coli
  • Support Protocol 3: Growing Phage Clones in Microtiter Plates for ELISA Testing
  • Basic Protocol 4: Phage ELISA
  • Support Protocol 4: Growing Soluble Fragments in Microtiter Plates
  • Basic Protocol 5: Soluble Fragment ELISA in Microtiter Plates
  • Basic Protocol 6: Amplification and Fingerprinting of Selected Clones
  • Support Protocol 5: Preparation of Periplasmic Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Concentration of Phage or Phagemid Particles by PEG Precipitation

  Materials
  • Culture of E. coli bearing the phage or phagemid—e.g., DH5αF′ (Life Technologies), TG1 (Stratagene): grown in recipe2× TY medium (unit 4.10) overnight at 30°C
  • 20% (w/v) PEG 6000/2.5 M NaCl
  • 10 mM Tris⋅Cl, pH 7.4 ( appendix 2A)/0.1 mM EDTA or 1× phosphate buffered saline (PBS; see recipe)
  • 0.45‐µm filter

Support Protocol 1: Preparing Helper Phage

  Materials
  • 2 × 1013 pfu/ml helper phage M13K07 (Pharmacia) or VCSM13 (Stratagene)
  • TE or recipePBS ( appendix 2A)
  • Male E. coli—e.g., DH5αF′ (Life Technologies Inc.), TG1 (Stratagene)
  • recipe2× TY medium (unit 4.10)
  • 2× TY top agar: add 6 g bacto agar/liter recipe2× TY medium; autoclave and allow to cool to 45°C
  • 2× TY plates (see recipe)
  • 5 mg/ml (1000×) kanamycin in water: filter sterilize and store in aliquots up to 1 year at −20°C
  • 60% (w/v) molecular‐biology‐grade glycerol, sterile
  • Toothpicks, sterile
  • 500‐ml culture flask
  • Additional reagents and equipment for monitoring bacterial growth ( appendix 1J), PEG precipitation (see protocol 1), rescuing phage particles (see protocol 3), and determining phage titer (see protocol 6)

Basic Protocol 2: Rescuing Phage/Phagemid Particles from Libraries

  Materials
  • Phage library:
  •  Peptide libraries (Mobitec or New England Biolabs) or
  •  Antibody libraries (obtain under collaborative agreements)
  • 2× TYAG: recipe2× TY medium (unit 4.10) containing 100 µg/ml ampicillin and 2% (w/v) glucose: store up to 1 month at room temperature
  • Helper phage (see protocol 2)
  • 2× TYAK: recipe2× TY medium containing 100 µg/ml ampicillin and 25 µg/ml kanamycin: store up to 1 month at room temperature
  • 30°C shaker incubator
  • 0.25‐ or 0.45‐µm filter
  • Additional reagents and equipment for monitoring cell growth ( appendix 1J) and PEG precipitation (see protocol 1)

Basic Protocol 3: Selection of Phage Antibodies to an Antigen Immobilized Indirectly or Directly on (Plastic) Surfaces

  Materials
  • 1 to 100 µg/ml antigen in carbonate buffer, pH 9.6 (unit 5.6), or PBS (see recipe)
  • PBS (see recipe)
  • MPBS: 2% and 4% (w/v) nonfat milk powder in recipePBS
  • 1–5 × 1012 PEG‐concentrated phage/ml (see protocol 1)
  • 0.1% (w/v) Tween 20 in recipePBS
  • 100 mM triethylamine (TEA), pH 12: Dissolve 140 µl triethylamine in 10 ml H 2O, prepare fresh
  • 1 M Tris⋅Cl, pH 7.4 ( appendix 2A)
  • 75 × 12–mm immunotube (Nunc)
  • Rotator
  • Additional reagents and equipment for determining phage titer (see protocol 6) and rescuing phage or phagemids (see protocol 3)

Alternate Protocol 1: Selection of Phage Antibodies Using Biotinylated Antigen and Streptavidin‐Paramagnetic Beads

  • Phage library:
  •  Peptide libraries (Mobitec or New England Biolabs)
  •  Antibody libraries (cannot be purchased, but can often be obtained under collaborative agreements)
  • Streptavidin paramagnetic beads (Dynal)
  • 100 to 500 nM biotinylated antigen: label using a commercial kit (e.g., Pierce); storage conditions are variable and antigen‐dependent
  • Dimethyl sulfoxide (DMSO; optional)
  • 2% (w/v) nonfat milk powder/0.1% (w/v) Tween 20 in recipePBS
  • PBS (see recipe)
  • 1 mM dithiothreitol (DTT)
  • End‐over‐end rotator
  • Magnetic rack (Dynal)
  • Additional reagents and equipment for determining viral titer (see protocol 6)

Support Protocol 2: Rescue of Phage/Phagemid by Infection of E. coli

  Materials
  • recipe2× TY medium (unit 4.10)
  • E. coli (Life Technologies)—i.e., single colony of TG1 on minimal agar or DH5αF′ in recipe2× TY medium
  • Phage/phagemid solution (see protocol 3 or protocol 5)
  • 90‐mm round and 14‐cm round or 243 × 243–mm square 2× TYAG plates (see recipe) containing appropriate antibiotics, dry
  • 2× TYAG medium: recipe2× TY medium (unit 4.10) containing 100 µg/ml ampicillin and 2% (w/v) glucose: store up to 1 month at room temperature
  • 60% (w/v) molecular‐biology‐grade glycerol
  • Microtiter plates (optional)
  • 30°C incubator
  • 15‐ and 50‐ml conical tubes
  • Spreader, sterile
  • Nunc Cryotube
  • Additional reagents and equipment for elution of phage (see protocol 3 and protocol 5) and monitoring cell growth ( appendix 1J)

Support Protocol 3: Growing Phage Clones in Microtiter Plates for ELISA Testing

  Materials
  • 2× TYAG medium (see protocol 3)
  • Bacterial colonies (i.e., antibody libraries and controls):
  •  Antibody library of interest—e.g., 2× TYAG plates (see recipe) containing output of selection round with evenly spaced colonies (see protocol 6)
  •  Uninfected E. coli (i.e., phage not produced)
  •  Phage not displaying antibody fragment
  •  Phage displaying nonrelevant antibody fragment
  •  Phage displaying antibody recognizing antigen of interest (positive control; optional)
  • 60% (w/v) molecular‐biology‐grade glycerol, sterile
  • 2 × 1013 pfu/ml helper phage (e.g., NEB, Stratagene; also see protocol 2) in recipe2× TY medium (unit 4.10)
  • 2× TYAK medium without glucose (see protocol 3)
  • 96‐well flat bottomed ELISA plates (e.g., Nunc maxisorb)
  • Toothpicks, sterile
  • 30°C shaker incubator and microtiter plate holder or enzyme plastic box (Boehringer) cushioned with foam
  • Adhesive tape
  • 96‐well transfer device (e.g., Boekel replicator)
  • 96‐well round‐bottom plate for bacterial culture (e.g., Nunc or Costar)
  • Tabletop centrifuge with microtiter plate carrier
  • Multichannel pipettor or suction device
  • Additional reagents and equipment for monitoring growth ( appendix 1J)

Basic Protocol 4: Phage ELISA

  Materials
  • 1 to 100 µg/ml protein antigen in carbonate buffer (unit 5.6) or PBS (see recipe)
  • PBS, pH 7.4 to 7.6 (see recipe)
  • MPBS: 2% and 4% (w/v) nonfat milk powder in recipePBS
  • 0.1% (w/v) Tween 20 in recipePBS
  • Culture supernatant containing phage antibody (see protocol 7)
  • Positive and negative controls (optional)
  • Horseradish peroxidase (HRP)–conjugated mouse anti‐phage mAb, (e.g., Pharmacia): dilute according to manufacturer's instructions
  • 10‐mg 2,2′‐azinobis(3‐ethylbenzthiazolinesulfonic acid) tablet (ABTS; Sigma)
  • 50 mM citrate buffer, pH 4.5 (see recipe)
  • 30% hydrogen peroxide
  • 0.02% sodium azide or 3.2 mg/ml sodium fluoride (ABTS developing)
  • TMB working solution (see recipe)
  • 2 N H 2SO 4 (TMB developing)
  • 96‐well ELISA plate, e.g., Nunc Maxisorb
  • ELISA plate reader (optional)

Support Protocol 4: Growing Soluble Fragments in Microtiter Plates

  Materials
  • 2× TYAG plates (see recipe) containing output of selection round with evenly spaced colonies (e.g., titration plates; see protocol 6)
  • Bacterial colonies with phage producing no antibody fragments and producing nonrelevant antibody fragments (controls)
  • 2× TYAG medium (see protocol 3)
  • 60% molecular biology grade glycerol, sterile
  • recipe2× TY medium (unit 4.10) containing 100 µg/ml ampicillin and 3 mM IPTG (diluted from a 1 M stock)
  • 96‐well flat‐bottomed microtiter plates (e.g., Nunc or Costar)
  • Toothpicks, sterile
  • 96‐well transfer device (e.g., Boekel replicator)
  • Tabletop centrifuge
  • Additional reagents and materials for monitoring cell growth ( appendix 1J)

Basic Protocol 5: Soluble Fragment ELISA in Microtiter Plates

  Materials
  • 1 to 100 µg/ml antigen in carbonate buffer (unit 5.6) or PBS (see recipe)
  • PBS (see recipe)
  • MPBS: 2% and 4% (w/v) nonfat milk powder in PBS
  • 0.1% (w/v) Tween 20 in recipePBS
  • Supernatants containing soluble antibody fragment (scFv), antibody which binds the coating antigen (positive control; optional), and nonbinding soluble antibody fragment (negative control; see protocol 9 for all supernatants)
  • 1.6 µg/ml mAb 9E10 (ATCC# CRL‐1729) in 2% MPBS
  • Horseradish peroxidase (HRP)–conjugated anti‐mouse secondary antibody: use according to manufacturer's specifications (e.g., Dako D260 is used at 1/1000 in 2% MPBS) or
  • Alkaline phosphatase (AP)–conjugated anti‐mouse secondary antibody: use according to manufacturer's specifications
  • 0.9% (w/v) NaCl (detection with alkaline phosphatase; optional)
  • 10‐mg ABTS substrate tablet
  • 50 mM citrate buffer, pH 4.5 (see recipe)
  • 30% (v/v) hydrogen peroxide
  • TMB solution, fresh (see recipe)
  • 20‐mg p‐nitrophenyl phosphate (pNPP) tablet
  • 20 ml pNPP buffer (see recipe)
  • 0.02% (w/v) sodium azide
  • 3.2 mg/ml sodium fluoride/ml in H 2O
  • 2 N H 2SO 4
  • 96‐well ELISA plate (e.g., Nunc Maxisorb)
  • Buffer tank

Basic Protocol 6: Amplification and Fingerprinting of Selected Clones

  Materials
  • 10× PCR buffer (provided by Taq manufacturer)
  • 5 mM dNTPs (see recipe)
  • 10 µM 5′ and 3′ primers
  • 5 U/µl Taq polymerase
  • 25 mM MgCl 2 (optional)
  • Single positive colony or bacterial growth identified by either phage ELISA (see protocol 8) or scFv ELISA (see protocol 10)
  • Purified recombinant yielding a similar sized fragment as the target in the same vector as investigated (positive control)
  • Light white mineral oil (Sigma; optional)
  • 2% agarose gel ( appendix 1N)
  • 10× TBE ( appendix 2A)
  • 10 mg/ml acetylated BSA
  • 10× BstNI buffer
  • 10 U/µl BstNI
  • Purified DNA known to contain BstNI sites
  • 4% Nuseive agarose (BMA) gel ( appendix 1N)
  • Ethidium bromide staining solution ( appendix 2A)
  • 10× Ficoll Orange: 0.4% (w/v) Orange G in 25% (w/v) Ficoll 400 in water
  • 100‐ to 500‐bp ladder
  • 96‐well PCR microplates (optional)
  • Toothpicks, sterile
  • UV transilluminator
  • Additional reagents and equipment for agarose gel electrophoresis ( appendix 1N)
CAUTION: Ethidium bromide, which is used to visualize the DNA, is a mutagen and should be handled with gloves and disposed of appropriately.NOTE: Many PCR materials are now supplied in kit format (e.g., NEB, Stratagene, Promega), which should be used as directed by the manufacturer.

Support Protocol 5: Preparation of Periplasmic Proteins

  Materials
  • Antibody library
  • 2× TYAG medium (see protocol 3)
  • recipe2× TY medium (unit 4.10) containing 100 µg/ml ampicillin and 0.1% (w/v) glucose
  • 2× TY medium containing 100 µg/ml ampicillin and 1 mM IPTG
  • 1 M IPTG: store in 100‐µl aliquots for up to a year or more at −20°C
  • 1 mM EDTA in PBS (see recipe for PBS), ice cold
  • 1 M MgCl 2
  • 250‐ml culture flask
  • 50‐ml conical tube
  • End‐over‐end rotator (optional)
  • 0.2‐µm filter or 10 kDa‐cut‐off dialysis tubing
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   De Bellis, D. and Schwartz, I. 1990. Regulated expression of foreign genes fused to lac: Control by glucose levels in growth medium. Nucl. Acids Res. 18:1311.
   de Haard, H.J., van Neer, N., Reurs, A., Hufton, S.E., Roovers, R.C., Henderikx, P., de Bruine, A.P., Arends, J.W., and Hoogenboom, H.R. 1999. A large non‐immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J. Biol. Chem. 274:18218‐18230.
   Evan, G.I., Lewis, G.K., Ramsay, G., and Bishop, J.M. 1985. Isolation of monoclonal antibodies specific for human c‐myc proto‐oncogene product. Mol. Cell Biol. 5:3610‐3616.
   Kay, B., Winter, J., and McCafferty, J. 1996. Phage Display of Peptides and Proteins. Academic Press, New York.
   Marks, J.D., Hoogenboom, H.R., Bonnert, T.P., McCafferty, J., Griffiths, A.D., and Winter, G. 1991. By‐passing immunization. Human antibodies from V‐gene libraries displayed on phage. J. Mol. Biol. 222:581‐597.
   Yamamoto, K.R., Alberts, B.M., Benzinger, R., Lawhorne, L., and Treiber, G. 1970. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to largescale virus purification. Virology 40:734‐744.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library