Phage - Displayed Antibody Libraries
Phage-displayed antibody libraries consist of large
repertoires of Fab fragments (Barbas et al.
, 1991), single
chain variable regions (scFv) (McCafferty et al.
or diabodies (dimer scFv) (McGuinness et al.
cloned into genetically engineered phage or phagemid
vectors (Smith, 1985; Smith and Petrenko, 1997), and
expressed on the surface of a bacteriophage. This
phenotype-genotype linkage enables phage-displayed
antibodies to be selected by multiple rounds of
antigen-based affinity purification and amplification.
In addition, phage-displayed libraries can be constructed
bypassing the immune system and therefore
can be targeted to self-antigens (Zeidel et al.
, 1995), as
well as to nonimmunogenic and even toxic substances
(Vaugham et al.
, 1996). Furthermore, being an in vitro
technique, phage display technology makes it easier to
build fully human antibody libraries (Holt et al.
This article illustrates a general protocol for the generation
of scFv libraries displayed on the surface of the
pCGMT phage vector as part of either surface protein
pIII (Gao et al.
, 1997; Mao et al.
, 1999; Gao et al.
or pIX (Gao et al.
II. MATERIALS AND
First-strand cDNA synthesis kit is from Amersham
Pharmacia (Cat. No. 27-9261-01). Carbenicillin, tetracycline,
kanamycin, and IPTG are from Research Products
International (Cat. No. C46000-1.0, T17000/1.0,
K22000-1.0, and I56000-5.0). Pfu
Escherichia coli XL-1 blue, VCSM13 interferenceresistant
helper phage, and total RNA purification kit
are from Stratagene (Cat. No. 6000154, 200249, 200251,
and 400790). Ultrapure agarose, electroporation
cuvettes, 1-kb plus DNA ladder, T4 DNA ligase, bovine
serum albumin (BSA), and 100 mM dNTP mix are from
Invitrogen (Cat. No. 15510027, 15224017, P450-50,
10787018, 15561012, and 10216018). Dimethyl sulfoxide
(DMSO) is from Aldrich (Cat. No. 27,043-1). SfiI is
from New England Biolabs (Cat. No. R0123S).
QIAquick gel extraction kit is from Qiagen (Cat. No.
28704). Tryptone is from Becton Dickinson (Cat. No.
211043). Yeast extract is from obtained EM Science
(Cat. No. 1.03753.5007). Nonfat dry milk is from Bio-
Rad (Cat. No. 170-6404). All the salts, glycerol, polyethylene
glycol, Tween 20, ethanol, and glucose are
from Sigma (Cat. No. G5516, P2139, P1379, 27,074-1,
G5767). Petri dishes and Nunc MaxiSorb Immunotubes
are from Fisher (Cat. No. 08-757-1000, and 12-
565-144). Polymerase chain reaction (PCR) tubes are
from USA Scientific (Cat. No. 1402-4300). Nalgene
500-ml centrifuge bottles are from VWR (Cat. No.
21020-050). The thermocycler (Mastercycler Gradient)
and benchtop centrifuge (5415C) are from Eppendorf.
The electrophoresis chamber (Easy-Cast) is from Electrophoresis
System. The electroporation apparatus
(Gene Pulser II) is from Bio-Rad. The J2-H2 centrifuge
is from Beckman.
A. Construction of a scFv Library
- 2xYT: To make 1 liter, dissolve 16 g tryptone, 10 g
yeast extract, and 5g NaCl in 900ml distilled water. After adjusting the pH to 7.0 with
NaOH, bring the volume to 1 liter. Sterilize by
- SB medium: To make 1 liter, dissolve 30g tryptone,
20 g yeast extract, and 10g MOPS in 1 liter deionized
water. Sterilize by autoclaving.
- LB-agar: To make 1 liter, dissolve 10 g tryptone, 5 g
yeast, and 10 g NaCl in 900 ml distilled water. Add
15 g agar and adjust the pH to 7.0 with NaOH. Fill
to 1 liter and autoclave. Allow to cool to a reasonable
temperature and supplement with 1 ml carbenicillin
stock, 10µg/ml tetracycline, and 2%
glucose. Pour in petri dishes.
- SOC: To make 1 liter, dissolve 20g tryptone, 5g
yeast, and 0.5 g NaCl in 900 ml distilled water. Add
10ml 250 mM KCl. Adjust the pH to 7.0, fill to 975
ml, and autoclave. Once cooled, add 5ml 2M MgCl2 and 20ml 1M glucose
- Phosphate-buffered saline (PBS): To make 1 liter, dissolve
1.44 g sodium phosphate, 0.24 g potassium
phosphate, 0.2 g potassium chloride, and 8 g NaCl
in 900ml distilled water. Adjust the pH to 7.4. Fill
to 1 liter and autoclave.
- Blotto: To make 100 ml, dissolve 4 g nonfat dry milk
in enough PBS to make 100ml final volume.
- 3 M NaOAc: Dissolve 24.61 g NaOAc in 90ml distilled
water. Adjust the pH to 5.2. Fill to 100 ml and
- 250 mM KCl: Dissolve 1.86 g KCl in 100 ml distilled
water. Sterilize by autoclaving.
- 2M MgCl2: Dissolve 40.7g magnesium chloride
hexahydrate in 100ml distilled water. Sterilize by
- 1M glucose: Dissolve 90g glucose in 500ml distilled
water. Sterile filter.
- Carbenicillin stock: Dissolve 1 g carbenicillin in
10ml deionized water. Sterile filter. Store at -20°C.
- Tetracycline stock: Dissolve 50mg tetracycline in
10ml 70% ethanol. Store at-20°C.
- Kanamycin stock: Dissolve 500mg kanamycin in
10ml deionized water. Sterile filter. Store at -20°C.
- 0.5M IPTG stock: Dissolve 5 g isopropyl-β-Dthiogalactopyranoside
(IPTG) in 42ml deionized
water sterile filter. Store at -20°C.
- VCSM13 helper phage solution: Prepare this solution
according to the vendor's instructions.
- Preparation of mRNA. Extract mRNA from either
human peripheral blood lymphocytes (human library)
or mouse spleen cells (murine library) using the RNA
Purification Kit (Stratagene) according to the vendor's
instructions. Prepare first-strand cDNA from the total
RNA by using the First-strand cDNA Synthesis Kit
(Pharmacia) and dT18 primer according to the manufacturer's
- PCR amplification of antibody variable region genes
(see Fig. 1). In a 250-µl PCR tube,
combine 2µl of cDNA template, 2µl of one forward
primer (100pmol/µl), 2µl of an equimolar mixture of
the respective reverse primers (100pmol/µl), 200µM dNTPs, 5% DMSO, 10 µl 10× Pfu DNA polymerase
buffer, and 5 units Pfu DNA polymerase (final volume:
100µl). For example, for the human heavy chain, set
up 12 PCR reactions total, in which each HVH forward
primer is combined with a mixture of HJH reverse
primers. Set the temperature program as follows:
denaturation at 94°C for 5 min; 30 cycles of amplification,
including denaturation, 1 min, 94°C; annealing,
1 min, 50°C; extension, 1 min, 72°C and final extension
at 72°C; for 10min. Run a 1% agarose gel and cut out
the appropriate bands. Combine the heavy chain
bands into one pool and the light chain bands (including
Vλ and Vκ) into a separate pool. Purify using a
Qiagen gel extraction kit.
- Construction of the scFv library (see Fig. 1). Assemble the scFv library by overlap PCR.
In a 250-µl PCR tube, combine ~20 ng of each scFv fragment
pool, 200µM dNTPs, 2µl 10× Pfu polymerase
buffer, and 1 unit Pfu polymerase (final volume: 20µl).
Set the temperature program as follows: denaturation
at 94°C for 5 min; 5 cycles of amplification, including
denaturation, 1 min, 94°C; annealing, 1 min; 55°C; and
extension, 1.5min, 72°C. Add the outer primers
HVH(Sfi) and HLJ(Sfi) [or MVH(Sfi) and MLJ(Sfi)] to a
final concentration of 2 mM and bring the final volume
to 50µl. Set the thermocycler for 30 more cycles of
denaturation, 30s at 94°C; annealing, 30s at 60°C; extension, 1.5 min at 72°C; and final extension at 72°C for 10min. Gel purify the full-length scFv library on a
1% agarose gel using a Qiagen gel extraction kit.
- Digestion of the scFv library and vector. Digest both
the scFv library and either pCGMT (pIII library; Gao et al., 1997) or pCGMT9 (pIX library; Gao et al., 2002) vector by combining 46 µl DNA, 6 µl 10x BSA, 6 µl 10x
NE buffer 2, and 2 µl SfiI. Incubate the mixture for 2h
or overnight at 50°C. Gel purify each digestion on a 1%
agarose gel using a Qiagen gel extraction kit.
- Ligation into vector. Combine 5µl SfiI digested
vector, 10 µl scFv library, 4 µl 5X ligase buffer, and 1 µl
T4 DNA ligase for a final volume of 20µl. Incubate at
16°C for 2-10h. Add 2µl of 3M NaOAc, pH 5.2, and
mix well. Add 40 µl absolute ethanol and store at -20°C for >20 min. Centrifuge at ≥12,000 rpm for 10 min.
Decant supernatant and wash the pellet with 1 ml 70%
ethanol, mixing well. Spin at≥12,000rpm for 1-2min
and aspirate the solution. Air dry the pellet and resuspend
in 10µl distilled water.
- Transformation into electrocompetent E. coli XL-1
Blue. Add 10µl of the ligation mixture to 500µl of a
suspension of E. coli XL-1 Blue. Add 50-µl aliquots to
10 prechilled electroporation cuvettes. Electroporate at
2.5 kV per manufacture's instructions. Immediately
add 200µl SOC medium to the cuvette and incubate at
37°C for 30min. Repeat as many times as necessary.
Plate the cells on LB-agar plates supplemented with
2% glucose, 50 µg/ml carbenicillin, and 10 µg/ml tetracycline.
Grow each plate overnight at 30°C.
- Library titer and storage. Scrape the clones off each
plate with 10 ml SB medium supplemented with 10%
glycerol and store at -70°C. Determine the size of the
library by counting the number of independent transformants.
To titer the library, serially dilute the library
(1:10) in SB out to 10-9. Starting with the lowest dilution,
add 100 µl onto a LB-carb plate and spread evenly.
Incubate overnight at 37°C and count the colonyforming
units for each dilution.
- Rescue of the scFv phage library. Inoculate ~5 × 105
cells oµained from the previously described glycerol
stock into 100ml SB medium containing 2% glucose,
50µg/ml carbenicillin, and 10µg/ml tetracycline.
Incubate the culture at 37°C on a reciprocal shaker
until OD600 ~ 0.6 is reached. Add ~4 × 1013 plaqueforming
units of VCSM13 helper phage and let incubate
for 30min at room temperature and then for
90 min on the shaker at 37°C. Add 200 µl of 0.5 M IPTG
and 140µl kanamycin and allow the culture to grow
over-night at 30°C
- Phage precipitation. Pellet the cells by centrifuging
at 7000rpm for 20min in a 400-ml centrifuge tube.
Decant the supernatant into clean tubes. Dissolve 3g
NaCl and 4 g PEG-8000 in each tube and place on ice
for 30 min. Centrifuge at 7000 rpm for 20 min to
pellet phage. Resuspend in an appropriate volume of
- Panning. Add 1 ml of ~5-50µg/ml antigen/
hapten to an immunotube and incubate overnight
at 4°C. Remove the antigen solution and block the tube
with 1 ml Blotto for 1 h at 37°C. Grow a culture of E. coli XL-1 Blue in 20 ml SB until the OD600 ~ 0.6. Save
for phage titration and rescue. Remove the blocking
solution and add 1012-1013 colony-forming units of
phage library in PBS containing 1% nonfat dry milk
and 3% BSA. Incubate at 37°C for 2h. Aspirate
unbound phage solution and remove weakly bound
phage by washing with 0.05% Tween 20 in PBS. Elute
the tightly bound phage by either adding 1 ml of 0.1M glycine, pH 2.5, or adding a solution of free antigen.
Incubate at room temperature for 10min. Remove the
solution and neutralize with concentrated Tris base
(usually 60µl 2M Tris, pH 8.0). Titrate the eluted phage
by serially diluting (1:10) in SB out to 10-9. Starting
with the lowest dilution, add 10µl diluted phage to
90µl E. coli XL-1 Blue. Plate 50µl onto a LB-carb plate
and spread evenly. Incubate overnight at 37°C and
count the colony-forming units for each dilution.
Rescue the remaining eluted phage by adding to 20ml E. coli XL-1 Blue, mixing gently, and letting sit for 10
minutes at room temperature. Centrifuge at 3000rpm
for 10 minutes and resuspend the pellet in approximately
500µ1 of the SB medium. Spread evenly on a
LB-carb plate and incubate overnight at 37°C Rescue
the library as outlined in step 7. This will be the input
library for the next round of panning. Phage libraries
are usually subjected to four to six rounds of panning.
The number of rounds is dependent on enrichment as
determined by library titer.
|FIGURE 1 scFv diagram indicating design and primer overlap.
HVH, human variable heavy chain; HJH, human constant heavy
chain; HVκ/λ, human variable light chain; HJκ/λ, human constant
light chain; HVH(Sfi), human forward SfiI primer; HLJ(Sfi), human
reverse SfiI primer.
This procedure outlines the construction of a human
or murine scFv library, including a list of suitable
primers. The technique is also applicable
to the formation of a Fab library with suitable
primers (Barbas et al.
The stringency of the phage selection can be
increased at the end of each round of panning in
several ways. The first is to decrease the amount of
antigen immobilized on the immunotubes. Similarly,
the incubation time of the library with the antigen
can be decreased. Alternatively, one can increase the
number of washing steps and/or the amount of detergent
in the wash buffer. Finally, one can use a progressively
lower concentration of free antigen during
the elution step. Note that when selecting for interactions
influenced by the ionic strength of the medium,
buffers other than PBS might be used for panning.
The number of transformations needed to obtain an
appropriate library size has to be determined experimentally.
Upon titration of the first transformation
mixture, 30-50 colonies must be picked and the corresponding plasmids digested to determine the percentage
of those containing inserts. Based on this number
and the overall titer, one can determine the number of
transformations needed to obtain ~109
The quality of a library depends not only on the
quantity of independent transformants, but also on
diversity. In order to test such diversity, a set of 30-50
random clones should be sequenced so that the percentage
of repeats can be determined. It is customary
to define a library as satisfactory if it contains less than
10% repeated sequences.
- Take extreme care when working with RNA to
prevent degradation by nucleases. This includes
wearing gloves and using nuclease-free solutions.
- Failure in the primary PCR reaction is typically due
to a low concentration of cDNA. Repeat the firststrand
synthesis if necessary. It may also be necessary
to adjust the annealing temperature. Note that
lowering the annealing temperature allows for less
fidelity in primer overlap.
- If overlap fails in step 3, check the concentrations of
the various VH, Vκ, and Vλ fragments by running a
small amount on a gel and examining the intensity
of the bands. Adjust the amounts accordingly to
give approximately equimolar concentrations.
- Scale the amount of phage preparation as necessary
for library size.
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