CaVHHLTM
Phage Display
Camel naïve VHH
Antibody Library Kit

User Manual

Cat. No. CaVHHTM
Published on Feb 10, 2012

CaVHHLTM: Phage Display Camel Single Domain Antibody Library

I. List of Components
CaVHHLTM: Phage Display Camel Single Domain Antibody Library. ~1.0×1012 recombinant phages

supplied in 100 μL PBS with 15% glycerol. Complexity=1.5×109 transformants; ampicillin
resistant.
E. coli TG1 host strain: K12 D(lac-pro), supE, thi, hsdD5/F’[traD36, proAB, lacIq, lacZDM15].
E. coli HB2151 host strain: K12 D(lac-pro), ara, nalr, thi/F’[proAB, lacIq, lacZDM15].
M13K07 helper phage: ~1.0×1011pfu phages (~1.0×1012 pfu/mL) supplied in 100μL TBS with 15% glycerol.
Kanamycin resistant.

The sequencing primers for CaVHHL Camel Single domain Antibody Library:TM

L1: 5'-TGGAATTGTGAGCGGATAACAATT-3'
S6: 5'-GTAAATGAATTTTCTGTATGAGG-3'２

CaVHHLTM: Phage Display Camel Single Domain Antibody Library

II. Introduction & Library Overview
Principle of phage display
In a phage display library, a variety of peptides, small antibodies [e.g. scFv and Fab] or proteins are
displayed on the surface of filamentous phage [M13, fd, and f1 strains] or lambda phage as fusion
proteins with one of the coat proteins of the phage virions, while the genetic materials encoding the
peptides/proteins are housed within the virion. Using a binding-based process called bio-panning [Figure1], a small number of phages that display proteins specifically binding to a target of interest can be
rescued from a phage library that usually displays a repertoire of many billions of uniquepeptides/proteins. Finally, the peptides/proteins displayed by the selected phages can be identified byphage amplification and DNA sequencing.

Figure 1. Illustration of phage display cycle

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CaVHHLTM: Phage Display Camel Single Domain Antibody Library

Figure 2. Phage display of antibody fragments.
The antibody fragment (such as variable heavy chain, VH, in red) is displayed as a fusion with theterminal phage geneIII protein (green). Both proteins are encoded by the phagmid DNA (magnified) and
expressed from a common promoter. The major gene VIII coat protein is shown in white (other phage
proteins not shown).

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CaVHHLTM: Phage Display Camel Single Domain Antibody Library

CaVHHLTM Phage Display Camel Single Domain Antibody Library
CaVHHLTM Phage Display Camel Single Domain Antibody Library was constructed by collecting antibody
gene pools from camel lymphocytes derived from blood samples. In brief, the VHH encoding genes wereinserted into pCDisplay-2TM vector, by which VHH (single domain variable fragment) antibodies werefused with phage coat protein III and displayed on the surface of phage virions. Using the protocol
provided in this manual, specific VHH binders can be selected very rapidly (generally within two months).
Using the protocol provided in this manual, specific single domain antibody binders can be selected very
rapidly (generally within two months). This library is an invaluable antibody resource for isolating camel
single domain antibody binders for research, diagnostic and therapeutic applications.

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CaVHHLTM: Phage Display Camel Single Domain Antibody Library

III. Materials and Methods for Using the LibraryMaterials
CaVHHLTM:

Phage Display Camel Single Domain Antibody Library, product of Creative Biolabs.

E. coli TG1 host strain: From Stratagene TG1 is a suppressor strain, in which the amber stop codon
would not be recognized and the single domain antibodies would be fused with phage coat protein III
and displayed on the surface of phage virions.
E. coli HB2151 host strain: From GE heathcare. HB2151 is a non-suppressor strain, in which the amberstop codon would be recognized and the soluble single domain antibodies would be produced.
M13K07 helper phage: GE Healthcare.

Media and solution
LB Medium: Per liter: 10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl. Autoclave 20 minutes at 121°C
LB Plates: LB medium + 15 g/L agarose
Top agar: as for LB medium, but use 7 g/L Bacto-agar. Before use, melt and cool to 50°C
2TY medium: 16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, made up to 1 L in deionized H2O, andadjusted to pH 7.0 with 1 M NaOH. Autoclave
2TY-G: 2TY containing 1%(w/v) glucose; 2TY-A: 2TY containing 100ug/mL ampicillin (AMP)
2TY-AG: 2TY containing 100ug/mL ampicillin (AMP) and 1%(w/v) glucose
2TY-AK: 2TY containing 100 ug/mL ampicillin, 50 ug/mL kanamycin
TYE agar plates: Add 15 g agar to 1 L 2TY medium, autoclave, when cool, add glucose to 1% (w/v) and
AMP
PBS: Per liter: 8 g NaCl, 0.2 g KCl, 1.7 g Na2HPO4, 0.163 g KH2PO4, pH to 7.4 with HCl
Blocking buffer: 0.1M NaHCO (pH 8.6), 5 mg/mL BSA, 0.02% NaN . Filter sterilize, store at 4°C
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Coating Buffer: 0.1M NaHCO3 (pH 8.6).
Conjugated second antibody:
HRP-conjugated anti-M13 monoclonal antibody from mouse: GE Healthcare
HRP conjugate of anti-c-Myc Tag: Pierce
EIA/RIA strip well 96-well plates: Corning, USA
Substrate: OPD and H2O2 from sigma
5*PEG/NaCl: Dissolve in water: 200 g of polyethylene glycol-8000, 150 g of NaCl(2.5M). Bring volume to
1 liter; stir until dissolved. Filter through a 0.8 µm filter
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CaVHHLTM: Phage Display Camel Single Domain Antibody Library

Library Screening
1. Maintenance of Bacterial Strains and Viruses
E. coli TG1 host strain: may be stored at 4 °C for up to two weeks on minimal medium; Prepare
glycerol stocks (add sterile 80% glycerol to stationary phase cells, mix well) store at -70°C for longterm storage.
E. coli HB2151 host strain: may be stored at 4 °C for up to two weeks on minimal medium; Prepare
glycerol stocks (add sterile 80% glycerol to stationary phase cells, mix well) store at -70°C for longterm storage.
M13K07 helper phage: This helper phage carries the kanamycin resistance gene for antibiotic
selection. Stocks of the phage can be stored at –80°C in 7% (v/v) DMSO indefinitely, or for up to 6
months at 4°C.
HuSdLTM: can be stored indefinitely at 4°C (in 1% BSA in PBS–0.01% [w/v] NaN3 ). Libraries should be
freshly amplified before panning. Keep plasmid DNA stocks of the primary library and/or glycerol
stocks of bacteria containing the phagemid library.

2. Amplification of M13KO7 helper phage1)

Prepare helper phage by infecting log-phase TG1 bacterial cells with M13K07 phage at different
dilutions for 30 min at 37°C and plating in top agar onto 2TY plates.

2)

Inoculate a small plaque in 3 mL liquid 2TY medium. Add 30 μL overnight culture of TG1 and growfor 2 h at 37°C.

3)

Dilute the culture in 1 L 2TY medium and grow for 1 h. Add kanamycin to 50μg/mL and grow for 16
h at 37°C.

4)

Remove cells by centrifugation (10 min at 5,000g) and precipitate phage from the supernatant byaddition of 0.25 vol of phage precipitant. After 30 min incubation on ice, collect the phage particles
by centrifugation during 10 min at 5,000g. Re-suspend the pellet in 5 mL PBS and sterilize through a
0.22-um filter.

5)

Titrate the helper phage by determining the number of plaque-forming units (pfu) on 2TY plateswith top-agar layers containing 100 μL TG1 (saturated culture) and various dilutions of phage.
Dilute the phage stock solution to 1 × 1013 pfu/mL and store in small aliquots at –20°C.

3. Panning
1)

Coat the target antigen (1-10μg/ml) in the panning plates in the coating buffer. Incubate at 37°C for
two hours.

2)

Block panning plates (NUNC) with the blocking buffer at 4 °C overnight.

3)

Wash blocked wells 6 times with 0.1%PBST (PBS with 0.1% Tween 20(V/V))4)

Mix equal volumes of the phage library and 4% PBSM (PBS containing 4% milk) in a total volume of７

CaVHHLTM: Phage Display Camel Single Domain Antibody Library
0.5 mL.
5)

During the first round of screening, the number of phage particles should be around 100× greaterthan the library size (e.g., 1012 pfu for a library of 1010 clones). Diversity drops to 106 after the firstround and thus there is no such a requirement in the subsequent rounds of screening. Incubate 30
min at room temperature to block the binding sites.

6)

Add input phage mix into panning wells and incubate at room temperature for 60 min.

7)

Wash 10-20 times with PBSMT (PBS containing 2% milk and a certain percent of Tween-20).

8)

Elute the phage by adding 200 μL either acidic eluting buffer or competitive eluting buffer andincubate 5 min at room temperature. Transfer the supernatant containing the phages to a new
tube.

9)

Infect a fresh exponentially growing culture of Escherichia coli TG1 with the eluted phages andamplify half of them for further rounds of selection.
a)

Inoculate 500 mL 2TY-G with the library glycerol stock and incubate at 37°C with shaking at
250 rpm until the optical density at 600 nm reaches 0.8–0.9.

b)

Add M13KO7 helper phage to a final concentration of 5 ×109 pfu/mL, and incubate for 30 min
at 37°C without shaking, then for 30 min with gentle shaking (200 rpm), to allow phage
infection.

c)

Recover the cells by centrifugation at 2,200g for 15 min and re-suspend the pellet in the same
volume of 2TY-AK. Incubate overnight at 30°C with rapid shaking (300 rpm).

d)

Pellet the cells by centrifugation at 7,000g for 15 min at 4°C and recover the supernatantcontaining the phage into pre-chilled 1-L bottles.

e)

Add 0.3 vol of phage precipitant. Mix gently and allow the phage to precipitate for 1 h on ice.

f)Pellet the phage by centrifuging twice at 7,000g for 15 min in the same bottle at 4°C. Remove
as much of the supernatant as possible and re-suspend the pellet in 8 mL PBS.

g)

Re-centrifuge the phage in smaller tubes at 12,000g for 10 min and recover the phage via thesupernatant. Ensure that any bacterial pellet that appears is left undisturbed.

h)Finally, titer phage stocks by infecting TG1 cells with dilutions of phage stock, plating to
2TY-AG, incubation, and enumeration of the numbers of ampicillin resistant colonies thatappear. The phages can then be stored in aliquots at 4°C for long periods, ready for screening.

4. Phage ELISA
1)

Prepare single clones of antibody-displaying phagesa)Inoculate single clones of the eulate from the 4th round into 5 mL of 2YT-AG medium and incubate
at 37°C overnight.

b)

Prepare the glycerol stock for each clone with the overnight culture. Inoculate 100μL of overnight
culture into 20mL of 2YT-AG medium. Grow for a few hours at 37°C until the optical density at

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CaVHHLTM: Phage Display Camel Single Domain Antibody Library
OD600 reach 0.4-0.5.
c)

Add M13KO7 helper phage at a multiplicity of infection of 20 (i.e., the number of phage
particles/host cell). Infect the cells by incubating 30 min at 37°C without shaking and another 30
min with shaking.

d)Collect infected cells by centrifugation (10 min at 5,000g). Re-suspend in 2YT-AK and grow theculture for 16 h at 30°C.

e)

Precipitate phage particles from the supernatant as described above. Re-suspend the phage pellet
in 1mL PBS and remove cellular debris by centrifugation (10 min at 5,000g).

f)

To remove Ab fragments not associated to phage particles, carry out a second precipitation.
Re-suspend the phage pellet in 250 μL PBS, clarify again by centrifugation.

2)

Phage ELISA by coating the target directlya)Coat 100 μL of non-biotinylated target (10μg/mL) in coating buffer by incubating at 4°C overnightb)

Shake out the coating solution and wash once with the washing buffer. Block all wells with 250 μLof blocking buffer. In order to test the binding of each selected sequence to BSA-coated plastic
surface, enough uncoated wells should also be blocked. Incubate the blocked plates 1-2 hours at
4°C.

c)

Shake out the blocking buffer and wash the plate 6 times with the washing buffer.

d)

Add 100 μL of phage solution in washing buffer per well. Incubate at room temperature for 1–2
hours.

e)

Wash 6 times with the washing buffer. Dilute HRP-conjugated anti-M13 antibody (GE healthcare)
1:5,000 in blocking buffer. Add 100 μL of diluted conjugate per well and incubate at room
temperature for 1 hour.

f)Wash 6 times with the washing buffer. Prepare the HRP substrate solution as follows: a stock
solution of OPD can be prepared in advance by dissolving 22 mg OPD (Sigma) in 100 mL of 50 mM
sodium citrate, pH 4.0. Filter sterilize and store at 4°C. Immediately prior to the detection step, add
36 μL 30% H2O2 to 21 mL of OPD stock solution.

g)

Add 100 μL of substrate solution per well and incubate at room temperature for 30 minutes.

h)Read plates using a microplate reader set at 490nm.

5. Soluble domain antibody ELISA
Since HB2151 is a non-suppressor strain, in which the amber stop codon between domain antibody geneand gene III can be recognized and the soluble domain antibody can be secreted into the periplasm.
1)

Soluble expression in periplasm of HB2151 cellsa)Pick a single colony of HB2151 from a LB plate and grow overnight at 37°C in 2TY.

b)

Add 100 μL HB2151 culture to 50 mL 2TY and grow at 37°C (with shaking) to an OD600 of 0.6–0.8.

９CaVHHLTM: Phage Display Camel Single Domain Antibody Libraryc)

Infect HB2151 with phages of positive clones identified by phage ELISA. Inoculate 1 μL of phages
into 200-μL aliquots of the log-phase HB2151 culture. Incubate for 30 min at 37°C (no shaking).
Plate onto TYE plates and incubate overnight at 37°C.

d)

Inoculate a single colony into 5mL aliquot of 2TY-AG growth medium and grow overnight at 37°C
(with gentle shaking).

e)Take a 250 μL aliquot of overnight culture from each well and transfer to 25 mL 2TY growth
medium. Grow the cultures at 37°C until OD600 is approx 0.6.

f)

Add IPTG to 1mM and continue to grow 20-24h at 30°C.

g)

Collect cells via centrifugation at 3,500g for 10 min. Release soluble domain antibody byultra-sonication of cell suspensions in 1/10 volume of PBS

h)Centrifuge at 9,000g for 10 min and collect the supernatant containing soluble domain antibody.

2)

Soluble domain antibody ELISAa)Coat the wells of an ELISA plate with the desired Ag (100 μL, 1-10μg/ mL) in coating buffer.

b)

Wash the wells 3× with 200 μL PBS.

c)

Block the wells with 200 μL PBSM for 2 h at room temperature.

d)Discard the block solution and add 100 μL of supernatant containing soluble domain antibody.
Incubate for 2 h at room temperature.

e)

Wash the wells 3× with 200 μL PBST.

f)

Add 100 μL of HRP conjugate of anti-c-Myc Tag Ab diluted in PBSM to each well and incubate for 1
h at room temperatureg)

Wash the wells 3× with 200 μL PBST.

h)Develop the HRP reaction using OPD and read optical density at 490nM

6.

DNA Sequencing

1)

Inoculate 2μL glycerol stock TG1 bacterial cells harboring the plasmid for each positive clone(determined by phage ELISA and/or soluble ELISA) into 5mL LB-A medium (LB medium added with
100μg/mL ampicillin). Grow overnight at 37°C with shaking.

2)

Isolate plasmids for each positive clone from bacteria using the commercialized Plasmid Isolation
Kit (Such as Qiagen Miniprep kit).

3)

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Conduct DNA sequencing using “L1” and “S6” as the primers.

CaVHHLTM: Phage Display Camel Single Domain Antibody Library

IV. Appendix:
1. Schematic map of pCDisplay-2TM(HuSdLTM was constructed by inserting VHH into pCDisplay-2TM)

2. One typical sequence of a single domain antibody:DNA:5’-gatgtgcagctggtggagtctgggggaggattagtgcagactggagggtctctgagactctcctgtgccgcctctggatacacacactgtaggtacg
acatggcttggtaccggcagactccagggaaggagcgcgagttcgtctcaagtattggtagggatggtagtacaacctacgcagcctccgtgaagggc
cgattcaccatctcccaagacaacaccaacaacacggtgtatctgctaatgagcagcctgagacctgaggacacggccatgtactactgcaagacattgt
ggtcggggatggaatgtactggtggtcactggggccaggggacccaggtcactgtctcctca-3’
AA:
CDR1
CDR2
DVQLVESGGGLVQTGGSLRLSCAASGYTHCRYDMAWYRQTPGKEREFVSSIGRDGSTTYAASVKCDR3GRFTISQDNTNNTVYLLMSSLRPEDTAMYYCKTLWSGMECTGGHWGQGTQVTVSS

References
1.
2.
3.

C. F. Barbas III, D. R. Burton, J. K. Scott, and G. J. Silverman, (2001) Phage Display: A laboratory
manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
O'Brien P.M. Aitken R. Totowa, (2001) Antibody phage display: methods and protocols. In: Methods
in Molecular Biology, Vol 178. Humana Press, NJ.
Sachdev S. Sidhu, (2005) Phage Display in Biotechnology and Drug Discovery. CRC Press/Taylor &１１

CaVHHLTM: Phage Display Camel Single Domain Antibody Library
Francis Group, Boca Raton.

Contact Information
For more information or technical assistance, please call, write, fax, or email to the following address:
Creative BioLabs
45-16 Ramsey Road
Shirley, NY 11967
USA
Tel: (631)-871-5806, Fax: (631)-207-8356
E-mail: info@Creative-Biolabs.Com
Web: www.creative-biolabs.com[© 2011 Creative BioLabs, CD Inc. All rights reserved]

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