27-9400 overview rev A 21/6/01 9:36 am Page 1

instructions

product code Recombinant Phage

27-9400-01
Antibody System
27-9401-01
27-9402-01 for cloning mouse antibody genes and
expressing and detecting functional antibodies.

Warning
For research use only.
Not recommended or
intended for diagnosis of
disease in humans or
animals.
Do not use internally or
externally in humans or
animals.

i 279400PL Rev-A, 2000

27-9400 overview rev A 21/6/01 9:36 am Page 2

Page finder
Handling
Components 2
Storage
The kits should be stored Safety warnings and precautions 3
according to the storage Description 4
instructions included with
individual modules Overview 5
Expiry
For expiry details see outer The Power of the technology 6
packaging of individual Immune response 8
modules
Antibody structure 10
Components M13 life cycle and vector systems 12
Mouse ScFv Module
Recombinant phage antibody system 25
Primed first-strand reaction Mouse ScFv Module 25
mixes (10): FPLCpure™
(Details in separate booklet)
Cloned Murine Reverse
Transcriptase, random Expression Module 29
hexadeoxyribonucleotides (Details in separate booklet)
[pd(N)6], RNAguard™, Detection Module 31
RNase/DNase-free BSA, (Details on Certificate of Analysis)
dATP, dCTP, dGTP, and Anti-E Tag Antibody 33
dUTP in aqueous buffer. (Details in separate booklet)
Control mRNA: Mouse
spleen mRNA in RNase- References 35
free water. Patent and licence information 37
DTT solution: 200 mM DTT
solution. Legal 40
RNase-free water: Treated Product information 40
with diethyl pyrocarbonate
(DEPC).
Heavy primer 1: 5' variable
heavy chain primer in
water (upstream primer).
Heavy primer 2: 3' variable
heavy chain primer in
water (downstream
primer).

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Light primer mix: Mixture of 10

Safety warnings and precautions
variable light chain primers
(both 5' and 3') in water. Warning: For research use only. Not
recommended or intended for diagnosis of
Linker-primer mix: Equimolar
mixture of heavy and light
disease in humans or animals. Do not use
chain linker primers in water. internally or externally in humans or
animals.
RS primer mix: Mixture of 5'
heavy chain primer with Sfi I
site and 3' light chain primers For detailed safety information relating to
with Not I site in water. individual modules please see the product
Sephacryl S-400 HR: Sephacryl instruction booklet for the appropriate
S-400 HR suspended in module.
distilled water and 20%
ethanol.
MicroSpin columns (25):
Polypropylene minicolumns,
each fitted with a
10 µm frit.
VH Marker: pUC 18/VH , EcoR
I/Xba I digested.
ScFv Marker: pUC 18/A10B, Sfi
I/Not I digested.

Expression Module
pCANTAB 5 E: Aqueous solution
(50 ng/µl) in TE buffer
(pH 7.6).
M13KO7 helper phage: Titred
phage in YT medium.
E. coli TG1 cells: Lyophilized.
E. coli HB2151 cells:
Lyophilized.
10× One-Phor-All buffer Plus:
Aqueous solution.
Control insert: A10B, Sfi I/Not I
digested.

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ScFv marker: pUC Description

18/A10B, Sfi I/Not I
digested. The Recombinant Phage Antibody System is
designed to clone mouse antibody genes and
Detection Module to express and detect functional antibodies. It
HRP/anti-M13 conjugate: is based on a phage-display system in which
Horseradish peroxidase
conjugated to sheep fragments of antibodies are expressed as
anti-M13 IgG (1:5 000 fusion proteins displayed on the phage
working dilution).
surface. When antibody genes are cloned in
Positive control phage:
M13KO7 bacteriophage. the phagemid vector pCANTAB 5 E, soluble
Positive control antigen: recombinant antibodies can also be produced
Sheep anti-M13 for use as immunological reagents. The
(10 µg/ml).
technology was developed in collaboration
ABTS™: 2',2'-Azino-Bis(3-
Ethylbenzthiazoline-6- with Cambridge Antibody Technology Ltd.,
Sulphonic Acid) UK (see important licensing information on
Diammonium (100 mg).
page 35).
The system is designed in a flexible four-
module format:
• Mouse ScFv Module
• Expression Module
• Detection Module
• Purification Module
The system includes sufficient reagents for
five separate recombinant phage antibody
libraries. Anti-E Tag Antibody is provided
separately for detection and purification of
soluble antibodies.
This booklet provides an overview of the
entire system. The protocols for the three
modules are provided separately.

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Overview
The Recombinant Phage Antibody System integrates fundamental con-
cepts and techniques from two very diverse fields of biology. To
understand the system requires that the researcher possess a basic
knowledge of the immunology and molecular biology involved. It is the
intent of this overview to provide this information. Five sections are
presented:
• The power of the technology (page 6)
• Immune response (page 8)
• Antibody structure (page 10)
• M13 life cycle and vector systems (page 12)
• Recombinant Phage Antibody System (page 25)
An extensive flowchart (starting on page 18) depicts the main steps of
the Recombinant Phage Antibody System and includes a timetable for
each phase.

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The power of the technology

Until recently, the production of antibodies was limited to very
laborious and time-consuming processes involving animal
immunization schemes and/or hybridoma generation. Recombinant
DNA and gene amplification technology, however, has now made it
possible to clone antibody genes in bacteria. Such 'immortalization' of
antibody genes makes it technically feasible to produce antibodies
quickly in bacterial cultures and to genetically manipulate their
structure such that ultimately, antibodies to any antigen may be
created.
One successful approach to recombinant antibody production has been
developed by McCafferty and coworkers (1, 2). This approach relies on
a phage-display system in which fragments of antibodies are expressed
as fusion proteins displayed on the phage surface. Using the polymerase
chain reaction (PCR*), immunoglobulin variable (V) region genes are
first amplified from hybridomas or spleen cells. The VH (variable
heavy) and VL (variable light) genes are then cloned into a phage vector
and expressed as a fusion with a phage protein. Each recombinant
phage genome contains the V genes that specify the antibody displayed
on its surface. Since the displayed antibody retains its antigen-binding
capability, it is thus possible to enrich for recombinant phage
expressing specific antibodies by affinity selection. With this approach,
antibodies of defined specificity and affinity can be selected from a
population.
Recombinant phage antibody technology has the power and versatility
to mimic the features of immune diversity and selection. In time, it may
even be possible to replace the current practices of animal
immunization and hybridoma development with a bacterial system
capable of synthesizing and expressing virtually unlimited quantities of
antibodies to practically any antigen.
Phage-displayed recombinant antibodies offer several advantages over
hybridoma-derived monoclonals:
• They are more easily cloned and screened. The cell fusion and
extensive screening required with monoclonal production are
time-consuming and labour-intensive processes. Specific antibody

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genes, however, can be cloned directly from spleen cells using rapid
and direct recombinant DNA methods. The time from isolation of
spleen cells to established antibody-producing clones is reduced
many-fold.
• They do not require animals or large-scale cell culture for their
production. Phage circumvent the need for ascites production or
large-scale cell culture. By generating a large natural display library
from V gene repertoires, it is even possible to completely bypass both
immunization and hybridoma development and isolate antibodies
with high affinity against any antigen (3). This is particularly
appealing in the area of human antibody development.
• They offer a stable genetic source. Some hybridomas are genetically
unstable and will lose or stop expressing antibody genes. Phage
antibody technology can be used to clone and rescue monoclonal
antibodies from unstable hybridomas (4).
• They can be genetically manipulated. Phage antibody genes can be
easily sequenced, mutated and screened to improve antigen binding.
It is evenpossible to rearrange the genes which code for the various
regions of an antibody molecule such that its specificity and affinity
for an antigen are altered (3).
* Purification using mouse monoclonal Anti-E tag as a ligand coupled
to Sepharose High performance offers a very specific purified
method.
Soluble recombinant antibodies add the following additional
advantages:
• They can be produced quickly and economically.
• They are useful as immunological reagents.
The power of recombinant phage antibodies, coupled with the ease
and versatility of recombinant DNA methodology, offer an improved
route for the production of highly specific reagents for research,
immunotherapeutic and diagnostic applications.

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Immune response
Antigens are substances that induce an immune response in an animal.
In general, they are polyvalent structures with numerous antigenic sites
or epitopes, which are recognized by cells that participate in the
immune response. B lymphocytes, which are located in the blood
(peripheral B lymphocytes), lymph nodes, spleen and several other
organs of the body, produce antibodies that specifically recognize and
interact with the antigen. These antibody molecules are encoded by
immunoglobulin genes in the B lymphocyte genome. When an animal is
challenged with a polyvalent antigen, a population of antibody-
producing B lymphocytes is stimulated. Each cell within the population
produces an antibody that recognizes a single epitope on the antigen.
The response is polyclonal since the collective population of antibodies
that develop will bind many different sites on the antigen. A single B
cell from this polyclonal population can be immortalized either by
mutation, fusion with myeloma cell lines (e.g. SP2/0-Ag14), or
transformation with Epstein Barr virus. The resulting clone secretes an
epitope-specific, or monoclonal antibody. Kohler and Milstein (5)
originally described the cell fusion and selection process for monoclonal
antibody production which is in widespread use today.

The Recombinant Phage Antibody System utilizes antibody-producing

hybridomas or B lymphocytes as a source of specific antibody genes. If
these cells are not producing antibodies, then the genes encoding the
antigen-specific antibody will be difficult or impossible to clone. To
verify that antibodies are being produced, the hybridoma culture
medium or the serum from the immunized animal should be assayed
for the presence of antigen-specific antibodies before attempting to
clone the specific immunoglobulin gene.

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Fv
Paratope CDR Heavy

;;;;;;;;;;;;;;;
NH2 Heavy Chain

;;;;;;; ;
Primers

;;;;;;;;;;;;;

;;;;;;;;;;;;;;;
;;;;;;;;;

;;;;;;; ;

;;;;;

;;
Light Chain

;;;

;;;;;

;;;;;

;;
COOH Light
Primers
CH
Idiotype
Fw

-SS
-SS
ScFv

-
-SS- CL Linker

;;;;;;;;;;;;;;;;
;;;;;;;;;;;
Isotype -SS- Heavy
Chain 5'

;;;;

; ; ;;
3' Light
Chain

Fd F(ab')2 Fab

;;;;;;;;;;

;;;;;;;;;;;;;;;;

;;;;;;;;;;;;;;;;
;;;;;;;;;;;

;;;;;;;;;;;
;;;;;

;;;;;;;;;;;
;;;;;;;;

;;;; ;

;;;;;;
;;;

; ; ;;

;;
;;

;
-

-SS

-SS
-SS

-
-SS-

Figure 1. Antibody model showing subunit composition and domain

distribution along the polypeptide chains. Fragments generated by
proteolytic cleavage and/or recombinant technology appear in the
shaded area.

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Antibody structure
Successful cloning of antibody genes requires some basic understanding
of antibody structure and function (6, 7). All antibodies have a
common structure (Figure 1, page 9) consisting of two identical heavy
(H) chain polypeptides (55–70 kd/440 amino acids) and two identical
light (L) chain polypeptides (24 kd/220 amino acids) held together by
disulphide bridges and non-covalent bonds. The four chains contain
defined Variable (V), Diversity (D) (heavy chain only), Joining (J) and
Constant (C) regions. The DNA and amino acid sequence of the C
region is relatively conserved within a given animal species while the V
region sequence is antigen-dependent. Pairing of the heavy and light
chain V regions creates an antigen-binding site (paratope) which
recognizes a single antigenic determinant (epitope). Most protein
antigens contain many potential epitopes which are recognized by a
correspondingly large number of specific antibodies that comprise a
polyclonal antibody population.

Constant region
The C region is located at the carboxy-terminus of an antibody chain.
In the mouse, there are five classes of constant heavy (CH) chain genes
(α, δ, ε, γ and µ) and two classes of constant light (CL) chain genes (κ
and λ). DNA and amino acid sequences are relatively conserved within
each class. Antibodies are grouped into five major classes according to
their CH region: IgA, IgD, IgE, IgG and IgM. An antibody is further
distinguished by the class of light chain
(κ or λ) paired with its heavy chain. The class of each chain can be
identified immunologically or isotyped with commercially available
reagents which are animal-species-specific.

Variable region
The V region is located at the amino-terminus of the antibody chain. It
consists of alternating framework (FW) and hypervariable, or
complementarity-determining regions (CDRs). The greatest sequence
diversity occurs in the CDRs, while the FW region sequences are more
conserved. The J region (heavy and light chains) and the D region

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(heavy chain only) lie immediately upstream from the C region. The
CDRs, and to some extent the FW regions, interact with the antigen to
form the core of an antigen-binding site. Sequence variations within the
J and D regions further influence the conformation of the site. The
cumulative effect of the variations in these regions determines the
antigenic affinity and specificity of an antibody, as well as its idiotype.
Because the DNA sequences within the first FW and the carboxy-
terminal portion of the J region are relatively conserved for all classes
of antibodies in a given animal, cloning becomes a feasible task.
Amplification schemes for antibody V genes can thus be devised using
a collection of primers that hybridize to these conserved sequences.
Since the Recombinant Phage Antibody System uses primers that
anneal to these conserved sequences, all classes of antibodies from
mouse can be amplified and cloned.
Antibody molecules also contain discrete protein domains or fragments
that can be isolated by protease digestion or produced by recombinant
techniques. Two functional antigen-binding fragments, designated Fab
and Fv, have been cloned and displayed on phage. The larger Fab
fragment (fragment antibody) contains the VL-CL and VH-CH segments
linked by disulphide bonds. The smaller Fv (fragment variable) is
composed of the VL and VH regions only. In a recombinant version of
the Fv fragment, designated the single-chain Fv fragment, or ScFv
(8-11), the two variable regions are artificially joined with a neutral
linker and expressed as a single polypeptide chain. This enables the VH
and VL regions to associate intramolecularly and stabilizes variable
domain combinations that interact weakly. Most VH-Linker-VL ScFv
proteins contain a single 15-amino acid linker (Gly4Ser)3. This flexible
peptide linker is specifically designed to bridge the 3.5 nm gap between
the carboxy-terminus of the VH chain and the amino-terminus of the
VL chain (8, 9). This construction facilitates chain pairing and
minimizes refolding and aggregation problems encountered when the
two chains are expressed individually. The affinity and stability of ScFv
antibodies containing the (Gly4Ser)3 residues are generally comparable
to those of the native antibody (8, 9, 12).

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H
Time
(hr)

Modules
L

-SS-

-SS-
-SS-

Cum.
Step
-SS-

Hybridoma/B cell

AAAA
Light chain mRNA

AAAA
Heavy chain mRNA

Day 1
1 1 mRNA Purification QuickPrep™ mRNA
Purification Kit

AAAA AAAA
Heavy chain (H) mRNA + Light chain (L) mRNA

Mouse ScFv Module

1 2 First-Strand cDNA Synthesis Reverse Transcriptase
+ pd(N)6

AAAA AAAA
TTTT TTTT
cDNA cDNA

3.5 5.5 Amplification 1 Primers

VH CH1 CH2 CH3 VL CL

AmpliTaq® + dNTPs
30 cycles
(Continued on page
(continued on page 21) 19)

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Amplification 1 (cont.)

Cum.
Step
Mouse ScFv Module (cont.)
VH VL

n n
Day 2
1 1 Gel Analysis
VH VL
300 bp —

1 2 Band Isolation
VH VL

n n

1 3 Gel Quantitation
VH VL
300 bp —

1.5 4.5 Assembly and

Fill-In Reaction
VH Linker VL

AmpliTaq® + dNTPs
7 cycles

VH Linker VL

(Continued
(continued onon page
page 22)20)

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Cum.
Primers

Mouse ScFv Module (cont.)

Step
Sfi I
3.5 8 Amplification 2 VH Linker VL Not I

AmpliTaq® + dNTPs
30 cycles

0.5 8.5 Spun-Column Purification

Sfi I VH Linker VL Not I

Day 3
1 1 Gel Quantitation

800 bp —

4 5 Sfi I Digest
+
Sfi I VH Linker VL Not I

4 9 Not I Digest
+ +
Sfi I VH Linker VL Not I
Day 4
0.5 0.5 Spun-Column Purification
Expression Module

2 2.5 Precipitation and

Gel Quantitation
800 bp —

(Continued
(continued onon page
page 23)21)

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Recombinant Phage Antibody System

The Recombinant Phage Antibody System was developed in a
collaboration between Amersham Biosciences and Cambridge
Antibody Technology Ltd., UK. Relevant background information
about cellular and molecular immunology, as well as phage cloning
systems, was summarized in the overview section. In the following
section, each step in the cloning process is described briefly. An
extensive flow chart is provided on pages 18–24. Detailed protocols are
provided with each module.
The Recombinant Phage Antibody System is designed in a flexible
modular format composed of three parts which are reviewed below:
• Mouse ScFv Module (below, page 25)
• Expression Module (page 29)
• Detection Module (page 31)

Mouse ScFv Module

Source of mRNA
Both mouse hybridomas and spleens are suitable sources of mRNA.
mRNA is an enriched source of expressed and properly spliced
antibody genes which can be isolated from either mouse antibody-
producing hybridomas, established cell lines or spleen-derived B
lymphocytes. Since a hybridoma expresses the heavy and light chain
genes for a single antibody, it therefore represents the most abundant
and straightforward source from which antibody genes can be cloned.
Phage antibodies can also be cloned using spleen cells from an
immunized mouse. However, since spleens express many different
antibody genes, more extensive selection and screening are required to
isolate a specific clone. In all cases, the antigen-binding activity of the
antibodies produced by either hybridomas or B lymphocytes should be
verified prior to cloning.

mRNA purification
mRNA is isolated from hybridoma or spleen cells. The success of
antibody cloning depends on the purity of the mRNA. QuickPrep™
mRNA Purification Kit is highly recommended for hybridomas since it

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provides high-quality mRNA, purified by affinity chromatography on

oligo(dT)-cellulose. While total RNA can be used, it is not
recommended since background products may also be amplified during
PCR. Any extraneous product will effectively decrease the yield and
relative purity of the desired PCR product, thus complicating the
cloning process. For spleens, isolation of total RNA followed by
affinity purification of mRNA on oligo(dT) cellulose is recommended
in order to effectively process the large number of cells present in a
spleen. RNA Extraction Kit and mRNA Purification Kit are highly
recommended.

First-strand cDNA synthesis

Reverse transcriptase synthesizes first-strand cDNA from mRNA
primed with random hexamers. The use of random hexamers
eliminates the need for immunoglobulin-specific primers or oligo(dT)
primers which would require the synthesis of a long cDNA, encoding
the entire heavy chain or light chain. Using random hexamers, the
resulting cDNAs are of sufficient length to clone the V regions from
both the heavy and light chain genes. Since these genes represent a very
small fraction of the total cDNA, they must first be amplified to
generate sufficient DNA for cloning.

Primary PCR
The heavy and light chain antibody genes are amplified in two separate
reactions. Two sets of primers, one set for each chain, are used in
separate amplification reactions. The primers are designed to hybridize
to opposite ends of the variable region of each chain. The heavy chain
amplification reaction yields a fragment approximately 340 base pairs
in length, while the corresponding light chain product is slightly
shorter, approximately 325 base pairs in length. The Recombinant
Phage Antibody System allows the cloning of a majority of the V region
gene families. Since lambda light chains represent only 5% of the
mouse light chain population, the sequence information available is
insufficient to allow consensus lambda-specific primers to be designed.
Therefore, lambda primers are not included with the kit, and lambda
light chain genes may not be amplified. If problems are encountered at

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this stage in the protocol, the light chain should be isotyped to

determine if the specific antibody bears a lambda light chain.

Gel purification
The amplified heavy and light chain fragments are purified separately
by agarose gel electrophoresis to remove primers and extraneous
amplification products. The Ethidium Bromide-stained, amplified DNA
bands are cut from the gel, extracted from the agarose plug and
purified. Purification of the DNA from an Agarose gel can be done
quickly and easily using the empty MicroSpin™ columns provided.
Alternatively, commercially available purification systems such as
Sephaglas™ BandPrep Kit (27-9285-01) may be used.

Gel quantitation of primary PCR products

The purified heavy and light chain DNA products are quantitated on
an agarose gel alongside a VH Marker of known concentration. The
amount of heavy and light chain and the amount of heavy chain
relative to the amount of light chain are quantitated so that the optimal
amount of each can be added to the assembly reaction.

Assembly of the heavy and light chains

The purified heavy and light chain DNA products are assembled into a
single gene using a DNA linker fragment. The linker fragment is
constructed such that one end anneals to the 3'-end of the heavy chain
while the other end hybridizes with the 5'-end of the light chain. The
assembly reaction ultimately produces a small amount of the single-
chain Fv gene where the VH region is linked to the VL region via a
sequence encoding a (Gly4Ser)3 linker which maintains the correct
reading frame. The ScFv DNA fragment is ~ 750 base pairs in length.

Second PCR amplification reaction

The assembled antibody ScFv DNA fragment is amplified with a set of
oligonucleotide primers that introduce restriction sites for cloning into
the pCANTAB 5 E vector. Sfi I and Not I sites are added to the 5'- and
3'-ends of the ScFv gene, respectively. These particular restriction sites
occur with very low frequency in antibody genes and should allow

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most ScFv genes to be cloned as a single Sfi I/Not I fragment.

Spun-column purification
The assembled antibody ScFv gene is purified on a spun column to
remove unincorporated linker primers and dNTPs. The components of
the polymerization reaction may interfere with subsequent steps if the
assembled ScFv DNA is not purified. In addition, the buffer is
exchanged in this step in preparation for the restriction digests.

Gel quantitation of purified ScFv fragment

The ScFv fragment is quantitated prior to restriction digestion. This
enables the determination of the number of units of each restriction
enzyme to add for optimal digestion of the ScFv product.

Restriction enzyme digestion and purification

The ScFv fragment is sequentially digested with Sfi I and Not I to
generate cohesive ends for ligation to the pCANTAB 5 E cloning vector.
The reaction is purified on a spun column to remove the small Not I
and Sfi I fragments that could interfere in the subsequent ligation
reaction. The cohesive ends generated in the restriction digests will be
used to clone the ScFv DNA fragment into pCANTAB 5 E (see
'Expression Module', on page 29 for additional details).

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References
1. McCafferty, J. et al., Nature 348, 552 (1990).
2. Winter, G. and Milstein, C., Nature 349, 293 (1991).
3. Marks, J. D. et al., J. Mol. Biol. 222, 581 (1991).
4. Carson, D. A. and Freimark, B. D., Adv. Immun. 38, 275 (1986).
5. Kohler, G. and Milstein, C., Nature 256, 495 (1975).
6. Roitt, I., Essential Immunology, Blackwell Scientific Publications,
London, seventh edition (1991).
7. Harlow, E. and Lane, D., Antibodies: A laboratory manual, Cold
Spring Harbor Laboratory, first edition (1988).
8. Huston, J. S. et al., Proc. Natl. Acad. Sci. USA 85, 5879 (1988).
9. Whitlow, M. and Filpula, D., Methods: A Companion to Methods
in Enzymol. 2, 97 (1991).
10. Batra, J. K. et al., J. Biol. Chem. 265, 15198 (1990).
11. Glockshuber, R. et al., Biochemistry 29, 1362 (1990).
12. Takkinen, K. et al., Protein Engineering 4, 837 (1991).
13. Rasched, I. and Oberer, E., Microbiol. Rev. 50, 401 (1986).
14. Messing, J., Gene 100, 3 (1991).
15. Vieira, J. and Messing, J., Methods in Enzymol. 153, 3 (1987).
16. Hoogenboom, H. R. et al., Nucl. Acids Res. 19, 4133 (1991).
17. Marks, J. D. et al., Bio/Technology 10, 779 (1992).
General Molecular Biology References
Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, second edition (1989).
Ausubel, F. M. et al., Current Protocols in Molecular Biology, Current
Protocols (1994).
Other General References
Innis, M. A. et al., PCR Protocols: A Guide to Methods and
Applications, Academic Press, Inc. (1990).
Borrebaeck, Carl A. K., Antibody Engineering: A Practical Guide,
W. H. Freeman Company (1992).

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Related products
Recombinant Phage Selection Module
1 kit 27-9403-01
RPAS Purification Module
1 kit 17-1362-01
Anti-E Tag Antibody
1 mg 27-9412-01
Anti-M13 Antibody
1 mg 27-9410-01
Light Primer Mix
10 µl 27-1583-01
Heavy Primers
10 µl each 27-1586-01
Linker Primer Mix
100 µl 27-1588-01
RS Primer Mix
100 µl 27-1589-01
pCANTAB 5 Gene Rescue Primers
1 nmol each 27-1581-01
pCANTAB 5 Sequencing Primer Set
250 pmol each 27-1585-01
QuickPrep mRNA Purification Kit
4 purifications 27-9254-01
RNA Extraction Kit
1 kit 27-9270-01
mRNA Purification Kit
2 purifications 27-9258-01
4 purifications 27-9258-02
Not I
200 units 27-0976-01
1000 units 27-0976-02
Sfi I
200 units 27-0975-01
1000 units 27-0975-02

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27-9400 overview rev A 21/6/01 9:36 am Page 37

Patent and licence information

Before using this product, carefully read the patent and licence information
below (see also the more detailed information supplied with the separate
modules of the Recombinant Phage Antibody System).
A licence for research use only of the technology which covers the phagemid
vector, antibody primers, and amplification method related to the
Recombinant Phage Antibody System, and the antibodies or molecules
derived therefrom, is granted by Cambridge Antibody Technology to the
end-user to use the technology, provided the Module has been purchased
from Amersham Biosciences. If you are in doubt as to whether your
use of antibodies or molecules derived from them would require a
commercial licence, please discuss the matter with Cambridge Antibody
Technology (see the instruction booklet for the Expression Module).
The purchase of an Amersham Biosciences Recombinant Phage
Antibody System/Mouse ScFv Module includes a fully paid-up, limited
licence under patent rights owned by Enzon Inc. granted to the end-user to
use these products for research use only. For information concerning the
availability of additional licences to utilize single-chain antigen-binding
molecules, contact Enzon Inc. (see the instruction booklet for the Mouse
ScFv Module).

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