Final scFv library construction involves the amplifi cation of VH and linker-VLDNA fragments from each cloned repertoire VH in pCANTAB6 and linker-VL in pCANTAB3his6, followed by assembl
Trang 18 Concentrate the digested VL repertoire by phenol/chloroform extraction, followed
by ethanol precipitation Gel-purify the large DNA fragment and estimate its concentration by comparison with markers
9 Perform suffi cient ligation reactions to ligate approx 0.4 µg dummy VH-linker DNA fragment into a 1-µg pool of the Vκ and Vλ libraries
10 Electroporate into E coli TG1 cells, and plate out as described previously (see
Subheading 3.2.) Aim to generate between 1 × 106 and 1 × 107 recombinants, carrying VL inserts with upstream scFv linker and dummy VH
3.4 Construction of the scFv Library (see Notes 8 and 9)
1 Amplify the VH and linker-VL DNA fragments separately from each of the cloned repertoires Perform 50 µL PCR reactions using the cycling parameters described
previously (see Subheading 3.2.), amplifying the VH repertoire with pUC19rev and JHFor primers and the VL repertoire with reverse JH and fdtetseq primers Purify the products from 1% TAE agarose gels and estimate DNA concentrations
by comparison with markers
2 Combine equal amounts of the VH and linker-VL PCR products (5–20 ng each), increase the total volume to 100 µL with ACS, reagent-grade H2O prior
to recovery of the DNA by ethanol precipitation Resuspend the DNA pellet
in 25 µL H2O
3 To perform the assembly reaction, add the following reagents to the pooled VHand linker-VL products, and perform 25 cycles of 94°C for 1 min, followed by 65°C for 4 min: 3.0 µL 10X Taq buffer, 1.5 µL 5 mM dNTP stock, and 0.5 µL
Taq polymerase (2.5 U).
4 Prepare 50 µL pull-through PCR reactions, pairing each VH BackSfiI primer with
either the Jκ1-5ForNotI primer mix or the Jλ1-5ForNotI primer mix Replicates of
each reaction are advisable, to maximize the diversity of the fi nal library Using 5.0 µL assembly DNA/reaction, amplify with cycling parameters described
previously (see Subheading 3.2.) The correct size of the assembled construct
is around 700 bp
5 Pool and concentrate the PCR products by phenol/chloroform extraction,
fol-lowed by ethanol precipitation Sequentially digest with SfiI and NotI restriction
endonucleases as described previously (see Subheadings 3.2 and 3.3.).
6 Gel-purify the digested scFv assembly construct and ligate with SfiI/NotI digested
pCANTAB6 after determining the optimum insert⬊vector ratio as described
previously (see Subheading 3.2.) Perform at least 100 electroporations, pool
into batches, and plate out each batch on large 243 × 243 mm 2TYAG plates Determine the total size of the library by taking aliquots from each batch and plating out serial dilutions on 2TYAG The fi nal library should contain in the region of 1 × 108 to 1 × 109 individual recombinants Clones picked from these
plates can be used to characterize the library (see Note 9).
7 Scrape the large plates, using 5 mL 2TY/plate, and pool the cells in 50-mL Falcon tubes Add 0.5 vol 50% (v/v) glycerol to each tube, and ensure homogeneous
68 Lennard
Trang 2resuspension of the cells by mixing on a rotating wheel for 30 min Determine cell density by optical density measurement at 600 nm Store the library in aliquots at –70°C.
3.5 Preparation of Library Phage (see Note 10)
1 Inoculate 500 mL 2TYG with 1010 cells from the library glycerol stock and incubate at 37°C with shaking at 250 rpm until the optical density at 600 nm reaches 0.5–1.0
2 Add M13KO7 helper phage to a fi nal 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
3 Recover the cells by centrifugation at 2200g for 15 min and resuspend the pellet
in the same volume of 2TYAK (2TY containing 100 µg/mL ampicillin, 50 µg/mLkanamycin) Incubate overnight at 30°C with rapid shaking (300 rpm)
4 Pellet the cells by centrifugation at 7000g for 15 min at 4°C and recover the
supernatant containing the phage into prechilled 1-L bottles
5 Add 0.3 vol of PEG/NaCl Mix gently and allow the phage to precipitate for
1 h on ice
6 Pellet the phage by twice centrifuging at 7000g for 15 min in the same bottle at
4°C Remove as much of the supernatant as possible and resuspend the pellet
in 8 mL TE buffer
7 Recentrifuge the phage in smaller tubes at 12,000g for 10 min and recover the
supernatant, which will now contain the phage Ensure that any bacterial pellet that appears is left undisturbed
8 Add 3.6 g of caesium chloride to the phage suspension and raise the total volume
to 9 mL with TE buffer Using an ultracentrifuge, spin the samples at 110,000g,
23°C, for at least 24 h
9 After ultracentrifugation, the phage should be visible as a tight band, which can be recovered by puncturing the tube with a 19-gage needle plus syringe and careful extraction
10 Dialyze the phage against two changes of 1 L TE at 4°C for 24 h
11 Finally, titer phage stocks by infecting TG1 cells with dilutions of phage stock, plating to 2TYAG, incubation, and enumeration of the numbers of ampicillin-resistant colonies that appear The phage can then be stored in aliquots at 4°C for
long periods (see Note 10), ready for screening (see Note 11).
4 Notes
1 Rapid processing of fresh tissue samples is essential if the full diversity of the
Ab repertoire is to be recovered If some loss of diversity is acceptable (perhaps when preparing libraries from infected or immunized individuals, rather than
in developing a comprehensive nạve library) tissue, mRNA, or cDNA product can be stored at –70°C
scFv Library Construction Protocols 69
Trang 32 RNA isolation (7) from Ficoll-isolated leukocytes, as described by Marks et
al (6), is the method of choice 50 mL of blood should yield approx 1 × 107cells, which in turn yield about 10 µg total RNA, of which 1–5% is mRNA It
is important to ensure that there is enough cDNA for all the VH and VL PCR reactions planned, each of which requires 0.5 ng cDNA
3 The PCR primers employed are based on those published by Marks et al (6),
and/or gene sequences in the V-BASE directory The 5′ and 3′ VH primers include
SfiI and XhoI restriction sites, respectively, to allow for cloning (see Table 1).
Include “no template” controls and check all PCR products on 1–2% (w/v) TAE agarose gels to ensure that a clean product of the expected size has been generated
4 Plasmid DNA (pCANTAB6 or pCANTAB3his6) is prepared either by the alkali lysis method (and subsequently caesium-banded as detailed in Sambrook
et al [8]), or by using a commercial kit (medium-scale) Approximately 20 µg
Cs-banded vector will yield ~5–10µg purifi ed cut vector Effi cient digestion with both enzymes is crucial to avoid self-ligation of the vector and high backgrounds
at transformation
5 A “vector only” ligation control should be included to determine the background caused by nonrecombinants Protocols for the preparation of electrocompetent
E coli TG1 cells and subsequent electroporations are described in Sambrook
et al (8) and by other contributors to this volume.
6 In most cases, a repertoire of ~1 × 107–1× 108 recombinants can be generated if 0.5µg digested VH segments are ligated with 1.5 µg digested vector
7 VLκ and VLλ gene fragments are amplifi ed separately using each back primer
in combination with the appropriate equimolar mixture of the Jκ or Jλ Forward
primers (see Table 2) After recovery of the combined VL repertoire, the next stage is to clone in the (Gly4Ser)3 scFv linker from an existing scFv, together with a dummy VH, recovered by PCR from an irrelevant clone Primer sequences
are shown in Table 3.
8 Final scFv library construction involves the amplifi cation of VH and linker-VLDNA fragments from each cloned repertoire (VH in pCANTAB6 and linker-VL
in pCANTAB3his6), followed by assembly on the JH region and amplifi cation
by pull-through PCR (see Table 3 for pull-through PCR primers) The resulting
scFv constructs (VH-linker-VL) are digested with SfiI and NotI and ligated into SfiI/NotI digested pCANTAB6.
9 Quality control analysis of the library is routinely performed by two methods to determine the percentage of recombinant clones and the level of library diversity For both methods, the fi rst stage is to PCR-amplify the scFv insert from 50 randomly picked clones/repertoire, using the vector primers, pUC19 reverse and
fdtetseq, as described in Subheading 3.2 Digestion of the PCR products with
BstNI restriction endonuclease and agarose gel electrophoresis can then be used
to visualize the restriction profi le for each clone The low cost and technical simplicity of this approach are its main strengths, but, as a means to assess the diversity of a library, it is limited by the resolving power of the agarose gel
70 Lennard
Trang 4Greater resolution and sensitivity can be achieved with polyacrylamide gels
and silver staining (8), but sequence analysis with fl uorescent dideoxy chain
terminators directly from the PCR products is clearly a better method, since it is
sensitive to single-base differences between clones beyond the BstNI recognition
sequence Each clone picked should carry a unique combination of VH and VLsequences
10 The resultant phage are purifi ed by PEG precipitation and caesium-banding, and,
as a result, are stable at 4°C for 2 yr Phage prepared by PEG precipitation alone should only be stored at 4°C for 1–2 wk
11 The affi nities of Abs directly isolated from scFv repertoires constructed in this manner without further engineering can be in the subnanomolar range and tend to have slower off-rates than those derived from rodent immune responses, smaller
scFv repertoires, or large synthetic Fab libraries (3).
References
1 McCafferty, J., Griffiths, A D., Winter, G., and Chiswell, D (1990) Phage
antibodies: fi lamentous phage displaying antibody variable domains Nature 348,
552–554
2 Winter, G., Griffi ths, A D., Hawkins, R E., and Hoogenboom, H R (1994)
Making antibodies by phage display technology Annu Rev Immunol 12,
agonist scFv Nature Biotechnol 15, 768–771.
5 Glover, D R (1999) Fully human antibodies come to fruition SCRIPS (May),
16–19
6 Marks, J D., Hoogenboom, H R., Bonnert, T P., McCafferty, J., Griffi ths, A D., and Winter, G (1991) By-passing immunization: human antibodies from V-gene
libraries displayed on phage J Mol Biol 222, 581–597.
7 Cathala, G., Savouret, J., Mendez, B., West, B L., Karin, M., Martial, J A., and Baxter, J D (1983) Method for isolation of intact, transcriptionally active
ribonucleic acid DNA 2, 329–335.
8 Sambrook, J., Fritsch, E F., and Maniatis, T (1990) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
scFv Library Construction Protocols 71
Trang 5From: Methods in Molecular Biology, vol 178: Antibody Phage Display: Methods and Protocols
Edited by: P M O’Brien and R Aitken © Humana Press Inc., Totowa, NJ
4
Broadening the Impact of Antibody Phage
Display Technology
Amplifi cation of Immunoglobulin Sequences
from Species Other than Humans or Mice
Philippa M O’Brien and Robert Aitken
1 Introduction
The production of monoclonal antibodies (MAb) through the tion of B lymphocytes has generally had little impact beyond human and murine immunology This can be explained by the lack of appropriate myeloma lines or transforming viruses for species outside this select group and the instability of heterohydridoma cell lines generated with, for example, murine
immortaliza-myeloma lines (1) The advent of Ab phage-display technology offers a
solution to this problem: success pivots upon the ability to recovery the immunoglobulin (Ig) repertoire from a source of B-lymphocyte mRNA and
to construct representative display libraries from the encoded proteins for screening In many species, understanding of the basis to Ig formation is now suffi ciently detailed for the application of these methods to MAb isolation.
We anticipate that the availability of MAb via phage display from a broad range of species will benefi t several areas:
1 To take livestock as an example, phage-display technology will obviate the modeling of viral, bacterial, or parasitic infections in rodent systems simply to obtain MAbs This should eliminate potential artifacts arising from the limited ability of many veterinary pathogens to colonize laboratory animals or differences
in antigenic recognition between natural and laboratory hosts
Impact of Antibody Phage Display Technology 73
Trang 62 In several important cases, human pathogens fail to establish in rodents, but relevant infection models are available in other animal species Similarly, there are many human diseases with close parallels in veterinary medicine The availability of MAbs from a wider range of species should increase the appeal
of animals other than rodents for the study of human disease The outbred characteristics of many of these mammals increases their value as models for human disease
3 If they are derived from the animal under investigation, passive transfer of MAbs should not provoke the antispecies responses triggered by delivery of murine monoclonals This may enable rapid evaluation of in vitro observations
in relevant animal infection models
4 The application of phage display should speed the development of MAb-based therapies for species of veterinary and economic importance and provide,
through transgenesis (2) or other novel methods of immunoprophylaxis (3), a
rational basis for enhanced disease resistance Other applications include passive immunomodulation of a range of physiological processes and Ig-targeted drug
or vaccine delivery (4).
To date, MAbs derived by phage display have been generated from rabbits
(5–7), chickens (8–10), sheep (11,12), cattle (13), camels (14), and primates (15–20) The Abs have been produced as scFv and Fab constructs, utilizing vec-
tors originally devised for human/murine immunology or expression systems optimized for the species under investigation Excluding rabbits and primates,
it is generally less complicated to amplify Ig-variable region sequences from veterinary species than from mice or humans Many domesticated species (e.g., cattle) predominantly express Ig λ light chains (LCs) compared to κ-chains, and, despite the apparent complexity of many LC loci, often the LC repertoire
is dominated by expression of a single or small numbers of families of
Vλ segments In addition, the expressed heavy-chain (HC) repertoire may be founded on single Ig HC gene families (e.g., cattle) or the rearrangement, diversifi cation, and expression of single HC or LC V segments (e.g., chick- ens) Overall this means that, in comparison to humans or mice, far fewer oligonucleotide primers are required to recover the Ig repertoire by polymerase chain reaction (PCR) from many of the species highlighted here.
This chapter presents a general protocol for the PCR amplification of expressed variable region sequences from a lymphoid RNA source and details oligonucleotide primers required for repertoire recovery from a selection of species other than mice and humans.
2 Materials
1 Purifi ed total RNA (peripheral blood, B-lymphocyte, lymphocyte-infi ltrated tissue, and so on) stored at –70°C
2 Sterile diethylpyrocarbonate-treated deionized H2O
74 O’Brien and Aitken
Trang 73 Maloney murine leukemia virus reverse transcriptase (MMLV-RT) and mercially supplied buffer(s).
4 10 mM Deoxyribonucleoside triphosphates (dNTPs), oligo(dT) primer, RNase
inhibitor
5 Taq polymerase and buffer (see Note 1).
6 Oligonucleotides for amplifi cation of species-specifi c Ig cDNA (see Tables
1–6 and Note 2).
7 Spin columns for cleanup of PCR reactions
8 10 mM Tris-HCl, 1 mM ethylene diamine tetraacetic acid, pH 7.4 (TE).
9 Ethidium bromide solution (2 mg/mL)
10 Stock of double-stranded DNA of defi ned concentration (e.g., determined by spectrophotometry) and 0.5–1 kb in size This can be generated by PCR or isolation of a restriction fragment from a plasmid
3 Methods
1 Aliquot 16 µM oligo (dT), 30 µg RNA, and the appropriate volume of
diethyl-pyrocarbonate-H2O to make a fi nal reaction volume of 100 µL (including the
reagents in step 2) into an RNase-free sterile microcentrifuge tube Heat at 70°C
for 10 min, then chill on ice
2 Add 200 U RNase inhibitor, buffer(s) to 1X fi nal concentration, 2 mM dNTPs,
and 500 U MMLV-RT Leave at room temperature for 10 min, then incubate at
37°C for 1 h (see Note 3).
3 PCR-amplify VH and VL sequences in a 100 µL reaction using 5 µL cDNA,
1X Taq polymerase buffer, 1.25–2.5 U Taq polymerase, 0.2 mM dNTPs, and
0.5 mM of each oligonucleotide primer (see Note 4) PCR conditions are 95°C for 5–15 min (see Notes 1 and 3), followed by 35 cycles at 95°C for
30 s, 52°C for 50 s, and 72°C for 1.5 min, followed by a fi nal incubation at 72°C for 10 min A separate PCR reaction should be performed for each primer combination
4 Check the amplifi cation of each Ig variable region by running a small aliquot of the reaction on a 1% agarose gel
5 Combine PCR reactions for each Ig class/isotype (VH, Vλ, Vκ) and clean up the reactions using spin columns
6 Gel-purify products on 1.5% agarose gels (see Note 5) and extract using spin
columns Check the purity of the PCR products by running on a second 1.5% agarose gel
7 Estimate the concentration of the products Prepare a series of dilutions of the isolated products in TE buffer Spot 5 µL to UV-transparent food wrap (e.g., plastic wrap) and set up a series of spots of a standardized DNA preparation Add equal volumes of ethidium bromide solution to each, and, by comparison
of fl uorescence intensities under UV illumination, calculate the concentrations
of the PCR products
(Text continues on page 83)
Impact of Antibody Phage Display Technology 75
Trang 10Targeted to C-terminal region of leader and framework Targeted to framework region 4 of VH
Trang 11Impact of Antibody Phage Displa
Trang 13Impact of Antibody Phage Displa
Trang 158 The amplifi ed products are now ready for restriction digestion or other modifi tions for insertion into the appropriate vector for expression as scFv or Fab
ca-(see Note 6).
4 Notes
1 In order to avoid nonspecifi c amplifi cation during PCR, it is best to use an amplifi cation protocol that incorporates a “hot start.” It is not advisable to use a polymerase that needs to be added to the tubes after denaturation of the template, because this increases the chance of contamination between samples There are many commercial options for enzymes that would be suitable: we fi nd that Hot
Star Taq polymerase (Qiagen, Germany) works well This enzyme requires a
15-min incubation at 95°C to become active
2 Oligonucleotide primers should be purifi ed before use in PCR to avoid cifi c amplifi cation and the recovery of truncated products Data in the tables are derived from the cited literature, but, for clarity and fl exibility, sequences encoding restriction sites, linker sequences, and so on, have been omitted Therefore, when designing primers, additional sequence should be added at the 5′ terminus of each primer to enable cloning of products into the phage-display vector selected for library construction, taking note of the reading frame(s) of coding regions fl anking the cloning site (e.g., bacterial leaders, purifi cation/detection tags, and so on) and adding standard linker sequences if scFvs are to be constructed by overlap extension prior to cloning All primers are shown 5′ to 3′ with standard codes for degeneracy The Ig reading frame and encoded amino acids are shown along with the region targeted by each primer Amino acids in brackets are encoded by the reverse complement of the primer sequence presented
3 Inactivation of RT is not necessary if a hot-start step is incorporated into the PCR method
4 The number of reactions required for recovery of the Ig repertore will depend
on the number of variable region families for the species of interest (see
Tables 1–6).
5 Do not overload the gels when purifying PCR products because contaminating PCR bands may be carried over, which can result in truncated products being incorporated preferentially into the expression vector
6 Some expression vectors have been modifi ed to express species-specifi c amino acid sequences around the VL and/or VH cloning sites (e.g., pComBov for
expression of bovine Fab [13]) If a general-purpose phage-display vector is to
be used, check its sequence and the amino acids encoded by restriction sites for the potential incorporation of nonnative residues at the termini of the mature
Ig fragment For example, if the vector adds murine sequences that differ from the residues commonly in the species under investigation, this may compromise the use of purifi ed MAbs in the host species at a later date
7 Specifi c Ig classes from camels (21) and llamas (22) are unusual, in that they
lack Ig LCs These Ig carry a single variable domain with amino substitutions Impact of Antibody Phage Display Technology 83
Trang 16at positions that would typically contact the LC-promoting interaction with the
solvent (23,24) They also lack the fi rst constant region domain Although this
sequence is present in the genome, it is spliced out during RNA processing
(25,26) Expression of these Ig in Saccharomyces cerevisiae is described in
Chapter 32
8 In several cases (15–20), libraries of primate Ig have been successfully contructed
with primers designed for recovery of the human repertoire For macaques, Table 6
shows primers used by Glamann et al (17) as an example of this approach For
chimpanzees, Table 6 shows only the species-specifi c primer targeted to the HC
hinge region (27), which was used with human primers by Schofi eld et al (19).
References
1 Suter, M (1992) The potential of molecular biology for the production of
monoclonal antibodies derived from outbred veterinary animals Vet Immunol
Immunopathol 33, 285–300.
2 Sola, I., Castilla, J., Pintado, B., Sanchez Morgado, J M., Whitelaw, C B A., Clark, A J and Enjuanes, L (1998) Transgenic mice secreting coronavirus
neutralizing antibodies into the milk J Virol 72, 3762–3772.
3 Lorenzen, N., Cupit, P M., Einer-Jensen, K., Lorenzen, E., Ahrens, P., Secombes,
C J., and Cunningham, C (2000) Immunoprophylaxis in fi sh by injection of
mouse antibody genes Nature Biotechnol 18, 1177–1180.
4 Wang, H., Griffi ths, M N., Burton, D R., and Ghazal, R (2000) Rapid body responses by low-dose, single-step, dendritic cell-targeted immunization
anti-J Immunol 96, 847–852.
5 Ridder, R., Schmitz, R., Legay, F., and Gram, H (1995) Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and
their production in the yeast Pichia pastoris Biotechnology 13, 255–260.
6 Foti, M., Granucci, F., Ricciardi-Castagnoli, P., Spreafi co, A., Ackermann, M., and Suter, M (1998) Rabbit monoclonal Fab derived from a phage display library
J Immunol Meth 213, 201–212.
7 Li, Y., Cockburn, W., Kilpatrick, J B., and Whitelam, G C (2000) High affi nity
scFvs froma single rabbit immunized with multiple haptens Biochem Biophys
9 Yamanaka, H I., Inoue, R., and Ikeda-Tanaka, O (1996) Chicken monoclonal
antibody isolated by a phage display system J Immunol 157, 1156–1162.
10 Cary, S P., Lee, J., Wagenknecht, R., and Silverman, G J (2000) Characterization
of superantigen-induced clonal deletion with a novel clan III-restricted avian monoclonal antibody: exploiting evolutionary distance to create antibodies specifi c for a conserved VH region surface J Immunol 164, 4730–4741.
84 O’Brien and Aitken
Trang 1711 Charlton, K A., Moyle, S., Porter, A J R., and Harris, W J (2000) Analysis of the diversity of a sheep antibody repertoire as revealed from a bacteriophage display
library J Immunol 164, 6221–6229.
12 Li, Y., Kilpatrick, J., and Whitelam, G C (2000) Sheep monoclonal antibody
fragments generated using a phage display system J Immunol Methods 236,
133–146
13 O’Brien, P M., Aitken, R., O’Neil, B W., and Campo, M S (1999) Generation of
native bovine MAbs by phage display Proc Natl Acad Sci USA 96, 640–645.
14 Arbabi Ghahroudi, M., Desmyter, A., Wyns, L., Hamers, R., and Muyldermans,
S (1997) Selection and identifi cation of single domain antibody fragments from
camel heavy-chain antibodies FEBS Lett 414, 521–526.
15 Samuelsson, A., Chiodi, F., Öhman, P., Putkonen, P., Norby, E., and Persson,
M A A (1995) Chimeric macaque/human Fab molecules neutralize simian
immunodefi ciency virus Virology 207, 495–502.
16 Tordsson, J., Abrahmsén, L., Kalland, T., Ljung, C., Ingvar, C., and Brodin, T
(1997) Effi cient selection of scFv antibody phage by absorption to in situ expressed
antigens in tissue sections J Immunol Methods 210, 11–23.
17 Glamann, J., Burton, D R., Parren, P W H I., et al (1998) Simian defi ciency virus (SIV) envelope-specifi c Fabs with high-level homologous neutral-izing activity: recovery from a long-term-nonprogressor SIV-infected macaque
immuno-J Virol 72, 585–592.
18 Siegel, D L., Reid, M E., Lee, H., and Blancher, A (1999) Production of large
repertoires of macaque MAbs to human RBCs using phage display Transfusion
21 Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., et al (1983)
Naturally-occurring antibodies devoid of light chains Nature 363, 446–448.
22 Vu, K B., Ghahroudi, M A., Wyns, L., and Muyldermans, S (1997) Comparison
of llama V-H sequences from conventional and heavy chain antibodies Mol
Immunol 34, 1121–1131.
23 Desmyter, A., Transue, T R., Ghahroudi, M A., et al (1996) Crystal structure of
a camel single-domain VH antibody fragment in complex with lysozyme Nature
Struct Biol 3, 803–811.
24 Spinelli, S., Frenken, L., Bourgeois, D., de Ron, L., Bos, W., Verrips, T., et al
(1996) The crystal structure of a llama heavy chain variable domain Nature
Struct Biol 3, 752–757.
Impact of Antibody Phage Display Technology 85
Trang 1825 Nguyen, V K., Muyldermans, S., and Hamers, R (1998) The specifi c variable
domain of camel heavy-chain antibodies is encoded in the germline J Mol Biol.
275, 413–418.
26 Woolven, B P., Frenken, L G J., van der Logt, P., and Nicholls, P J (1999) The structure of the llama heavy chain constant genes reveals a mechanism for
heavy-chain antibody formation Immunogenet 50, 98–101.
27 Ehrlich, P H., Moustafa, Z A., and Ostberg, L (1991) Nucleotide sequence of
chimpanzee Fc and hinge regions Mol Immunol 28, 319–322.
86 O’Brien and Aitken
Trang 19From: Methods in Molecular Biology, vol 178: Antibody Phage Display: Methods and Protocols
Edited by: P M O’Brien and R Aitken © Humana Press Inc., Totowa, NJ
recognize a variety of Ags (1,2) The rearranged V genes were amplifi ed
with the polymerase chain reaction (PCR) from B-cell mRNAs encoding immunoglobulin M (IgM) taken from nonimmunized donors By using this procedure, Abs were recovered prior to encounter with Ag and unscreened for tolerance by the immune system Indeed, a nạve library represents a good source of Abs against self, nonimmunogenic, and toxic Ags if the library is suffi ciently large and diverse.
Library size is a major determinant in successful selection against a large
set of Ags and it also correlates with the affi nity of the isolated Abs (3) Only
Abs with moderate affi nities were selected from the fi rst small libraries, but,
by increasing the repertoire size in the construction of later libraries, Abs with
better affi nities have since been obtained It has also been established (3,4)
that larger libraries deliver greater numbers of different Abs against target