Báo cáo khoa học: Selection of full-length IgGs by tandem display on filamentous phage particles and Escherichia coli fluorescence-activated cell sorting screening doc

Selection of full-length IgGs by tandem display on
filamentous phage particles and Escherichia coli
fluorescence-activated cell sorting screening
Yariv Mazor
1,2
, Thomas Van Blarcom
1,2
, Sean Carroll
1,2
and George Georgiou
1,2
1 Institute for Cellular and Molecular Biology, University of Texas at Austin, TX, USA
2 Department of Chemical Engineering, University of Texas at Austin, TX, USA
Introduction
Recombinant antibodies have made a tremendous
impact on biomedical research, and are increasingly
being used as clinical diagnostic and therapeutic
reagents [1,2]. Consequently, the demand for new tech-
nologies that aid in the discovery and selection of
novel therapeutic antibodies has never been greater.
During the past two decades, several display technolo-
gies and other library screening techniques have been
developed for the isolation of antigen-specific antibod-
ies from large collections of recombinant antibody
genes [3]. Phage display is the most prevalent method
for the display of large ensembles of antibody frag-
ments, and is currently considered to be the standard
procedure in many molecular biology laboratories for
antibody discovery and evolution [3]. Unique antibod-
ies are isolated from immune [4–7], naı
¨

ve [8–13] or
synthetic [14–21] repertoires, and are further engi-
neered for improved affinities for their antigens by
using the selected antibody gene as the basis for subse-
quent libraries and screening [22–26]. Humira [27,28]
Keywords
fluorescence-activated cell sorting (FACS);
full-length IgG; fUSE5–ZZ phage; protective
antigen (PA); spheroplasts
Correspondence
G. Georgiou, Department of Chemical
Engineering, University of Texas at Austin,
Austin, TX 78712, USA
Fax: +1 512 471 7963
Tel: +1 512 471 6975
E-mail:
(Received 7 February 2009, Revised 4
March 2010, accepted 9 March 2010)
doi:10.1111/j.1742-4658.2010.07645.x
Phage display of antibody libraries is a powerful tool for antibody discov-
ery and evolution. Recombinant antibodies have been displayed on phage
particles as scFvs or Fabs, and more recently as bivalent F(ab¢)
2
.We
recently developed a technology (E-clonal) for screening of combinatorial
IgG libraries using bacterial periplasmic display and selection by fluores-
cence-activated cell sorting (FACS) [Mazor Y et al. (2007) Nat Biotechnol
25, 563–565]. Although, as a single-cell analysis technique, FACS is very
powerful, especially for the isolation of high-affinity binders, even with
state of the art instrumentation the screening of libraries with diversity

>10
8
is technically challenging. We report here a system that takes advan-
tage of display of full-length IgGs on filamentous phage particles as a
prescreening step to reduce library size and enable subsequent rounds of
FACS screening in Escherichia coli. For the establishment of an IgG phage
display system, we utilized phagemid-encoded IgG with the fUSE5–ZZ
phage as a helper phage. These phage particles display the Fc-binding ZZ
protein on all copies of the phage p3 coat protein, and are exploited as
both helper phages and anchoring surfaces for the soluble IgG. We demon-
strate that tandem phage selection followed by FACS allows the selection
of a highly diversified profile of binders from antibody libraries without un-
dersampling, and at the same time capitalizes on the advantages of FACS
for real-time monitoring and optimization of the screening process.
Abbreviations
CFU, colony-forming units; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; IPTG,
isopropyl thio-b-
D-galactoside; PA, protective antigen; RU, response units; V
H
, variable heavy; V
L
, variable light.
FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS 2291
was the first fully human mAb discovered using phage
display to receive FDA approval, and at least 16
human mAbs derived from phage display are currently
in advanced clinical trials for a wide range of human
diseases [29,30].
Phage display is based on encoding the gene of
interest in-frame with one of the phage coat proteins

(phenotype), and encapsulates the fusion gene within
the phage particle (genotype). Recombinant antibodies
have been displayed on phage particles as scFv [31] or
Fab [7,32] fragments. These monovalent proteins,
although relatively easy to produce in Escherichia coli,
are typically devoid of avidity effects that allow the
recovery of low-affinity binders [33–36]. Polyvalent
display of scFv and Fab can be readily achieved, par-
ticularly by using phage-based vectors [37]. The resul-
tant avidity effects allow for the recovery of low
affinity-binders, but these same avidity effects make it
difficult to select stringently on the basis of intrinsic
affinity.
More recently, bivalent Fab [F(ab¢)
2
] has been dis-
played on phage particles in a manner that effec-
tively resembles the binding behavior of natural IgGs
[36]. Nevertheless, for the vast majority of diagnostic
and therapeutic applications, antibody fragments iso-
lated from most existing display technologies must
be converted to full-length IgG, the format of choice
in the clinics. This process requires additional cloning
steps and the expression of the reformatted antibody
gene in mammalian cells. A conspicuous drawback
of the scFv format is that reformatting to IgG can
result in loss of activity (TVB, YM, SAC, SK, BLI
and GG, unpublished observations). Yet another dis-
advantage of most existing phage display systems is
that the antibody gene is expressed as a fusion pro-

tein with one of the phage coat proteins. As a result,
many of the antibodies isolated through library
screening can only fold in the context of a fusion
protein and cannot be expressed independently, a
phenomenon that many laboratories do not report
[38].
We recently reported the development of an E. coli-
based technology, termed E-clonal, for the successful
production of soluble full-length IgGs in bacteria and
for screening of combinatorial IgG libraries using bac-
terial periplasmic display [39,40]. Library cells express-
ing intact IgGs specifically labeled with fluorescently
conjugated antigen are readily distinguished and iso-
lated by fluorescence-activated cell sorting (FACS).
Unlike phage display, FACS has the distinct advantage
of relying on real-time quantitative multiparameter
analysis of individual cells, allowing single-cell resolu-
tion for selection. Although FACS is a very powerful
high-throughput screening methodology, sorting a
library > 10
9
cells using FACS is time-consuming and
challenging [40–43].
To reduce the initial library to a size that is manage-
able by FACS and to demonstrate that full-length IgG
libraries can be displayed on phage particles and
undergo selection, we sought to develop a display sys-
tem that will effectively display intact IgGs on filamen-
tous phage particles. The system can efficiently
downsize very large libraries by employing an initial

round of phage biopanning that specifically pre-
enriches target cells from the library prior to subse-
quent rounds of FACS. Specifcally, E. coli ⁄ F¢ cells
expressing soluble IgGs in the periplasm (E-clonal
cells) are simultaneously infected with the fUSE5–ZZ
phage [44]. These phage particles allow polyvalent dis-
play of the Fc-binding ZZ protein [45] on all five cop-
ies of the gene-3 minor coat protein of filamentous
bacteriophages [44]. The fUSE5–ZZ phage in this sys-
tem serves not only as a helper phage, but also as the
IgG-capturing surface via the surface-displayed ZZ
protein (Fig. 1). Rescued fUSE5–ZZ–IgG phage parti-
cles harboring the IgG phagemid are then selected for
antigen binding by standard phage biopanning. We
describe here the feasibility of propagating phage parti-
cles that stably display functional full-length IgGs, and
demonstrate that an initial round of phage biopanning
followed by FACS facilitates the isolation of a diversi-
fied collection of antigen-specific binders from very
large antibody libraries.
Results
Model system validation
As a model for our studies and for validation of the
display of full-length IgGs on phage particles, we
chose two well-characterized antibodies, M18 and
26.10, which are specific for the protective antigen
(PA) from Bacillus anthracis (K
d
=30pm) and digoxin
(K

d
= 1.7 nm), respectively [39]. The full-length heavy
and light chain genes were expressed from a dicistronic
operon and secreted into the periplasm, where they
assembled into aglycosylated IgGs that were fully func-
tional for antigen binding [39]. To display the full-
length IgG noncovalently on phage particles, we uti-
lized fUSE5–ZZ, which displays the Fc-binding ZZ
protein [45] on all five copies of the gene-3 minor coat
protein, but maintains its ability to infect and propa-
gate in E. coli ⁄ F¢ cells [44] (Fig. 1). As the pMAZ360–
IgG expression vector contains the packaging signal of
f1 bacteriophage that enables the packaging of the
plasmid as ssDNA in the presence of a helper phage,
Selection of IgG by tandem phage panning-FACS Y. Mazor et al.
2292 FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS
rescued fUSE5–ZZ–IgG particles harbor the high copy
number pMAZ360–IgG phagemid preferentially over
the very low copy number and replication-defective
fUSE5–ZZ genome [44].
Initially, fUSE5–ZZ particles were evaluated for
their ability to capture purified IgGs in solution. The
phage particles were mixed with purified M18 IgG or
26.10 IgG, washed to remove any unbound antibodies,
and analyzed by ELISA. Phage that had captured the
purified IgG via the ZZ protein showed strong ELISA
signals with the respective antigens but not with unre-
lated antigens (data not shown). We then evaluated
whether fUSE5–ZZ is able to capture IgG within the
periplasm and form a noncovalent complex that is sta-

ble upon extrusion of the phage from the bacteria.
E. coli K91K ⁄ F¢ cells transformed with phagemid
pMAZ360–M18–IgG or pMAZ360–26.10–IgG were
grown under conditions permissive for phage infection.
Following infection with fUSE5–ZZ, the cultures were
allowed to grow overnight under conditions favorable
for phage production. On the following day, phage
particles were precipitated and evaluated for specific
binding by direct ELISA (Fig. 2). As expected,
fUSE5–ZZ propagated in cells expressing M18 IgG
bound specifically to PA (Fig. 2A), whereas phage par-
ticles produced in cells expressing 26.10 IgG bound
specifically to digoxin (Fig. 2B). Competition of the
bound IgG by an excess of standard human IgGs
resulted in a small reduction ( 15%) of ELISA signal
(Fig. 2A,B), indicating that the phage ZZ–IgG com-
plex is kinetically very stable, presumably owing to the
polyvalent display of the ZZ protein on all copies of
the phage p3 coat protein.
To determine the number of IgG molecules dis-
played on fUSE5-ZZ–IgG particles, we employed the
technique described by Junutula et al. [46]. Purified
fUSE5-ZZ–M18 IgG phage was applied at varying
concentrations to ELISA plates coated with anti-M13
IgG, anti-Fc IgG, or PA. Following incubation, the
ELISA was developed with horseradish peroxidase
(HRP)-conjugated anti-M13 IgG. The number of IgG
molecules displayed on each phage particle was deter-
mined by the ratio of the linear range of the ELISA
signals obtained with anti-Fc IgG ⁄ anti-M13 IgG or

PA ⁄ anti-M13 IgG (Fig. 2C). Analysis of the results
indicated that there is an average of 0.6–0.7 IgG mole-
cules per phage particle.
To assess the efficacy of the IgG phage display sys-
tem for selections, we tested the ability to enrich
fUSE5–ZZ–M18 IgG phage particles from a
1 000 000-fold excess of phage particles displaying the
control 26.10 IgG. The mixture was subjected to three
rounds of phage biopanning against PA, and the
enrichment after each round of selection was moni-
tored by ELISA (Fig. 2D). The increase in ELISA sig-
nal for PA in parallel with the decrease in ELISA
signal for digoxin clearly indicated a significant enrich-
ment of the fUSE5–ZZ–M18 IgG phage population at
the expense of a reduction in the number of phage par-
ticles displaying the control 26.10 IgG. Sequence anal-
ysis revealed that, following the third round of phage
panning, seven of 20 randomly selected clones carried
+
fUSE5-ZZ
g3
ZZZZ
V
L
C
L
P
pMAZ360-IgG
V
H

C
H1
C
H2
C
H3
pelB pelB
PLAC
Fd ori
Tet
R
Amp
R
F1ori
pMAZ360-IgG
Fig. 1. Schematic diagram of the IgG phage display format. Left: map of phagemid pMAZ360–IgG for expression of soluble intact IgGs in
the E. coli periplasm. This vector facilitates convenient cloning of V
H
and Vj domains linked to human c1 and j constant domains, respec-
tively, as a bicistronic operon downstream of the lac promoter. Center: map of fUSE5–ZZ phage for polyvalent display of the Fc-binding ZZ
protein on all copies of the gene-3 minor coat protein. Right: infection of E. coli cells carrying phagemid pMAZ360–IgG with fUSE5–ZZ leads
to the production of fUSE5–ZZ–IgG phage particles that stably display functional full-length IgGs.
Y. Mazor et al. Selection of IgG by tandem phage panning-FACS
FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS 2293
the M18 mAb sequence, corresponding to over
300 000-fold enrichment.
Library selection by tandem phage biopanning
and FACS
To evaluate the utility of tandem phage panning–
FACS for library screening, we used an anti-PA mouse

immune IgG library [39]. This library was constructed
by cloning pools of variable heavy (V
H
) and variable
light (V
L
) genes from spleens of mice that were immu-
nized with PA from B. anthracis into the pMAZ360–
IgG vector. Library DNA was transformed into
E. coli K91K ⁄ F¢ to generate a total of 10
7
independent
transformants. Cells carrying phagemid pMAZ360–
IgG were infected with fUSE5–ZZ, and the culture
was allowed to grow under conditions favorable for
phage production. Phage particles were purified and
subjected to an initial cycle of panning by incubation
with soluble biotinylated PA in solution, before being
captured on streptavidin-coated magnetic beads.
Unbound phage particles were removed by washing,
and bound phage particles were eluted, neutralized,
and used for infection of E. coli Jude-1⁄ F¢ cells har-
boring plasmid pBAD33–NlpA–ZZ for subsequent
rounds of FACS screening in cells expressing an
NlpA–ZZ fusion that allows capture of the IgG on the
inner membrane. Cells were converted to spheroplasts
by disruption of the outer membrane with Tris ⁄ EDTA
and lysozyme treatment, to allow exposure of the
membrane-bound IgG to the extracellular fluid. Two
color flow cytometry steps, using PA63–fluorescein iso-

thiocyanate (FITC) and Alexa Fluor 647–anti-(human
IgG) to monitor for affinity and expression of full-
length IgG, were employed, and fluorescent clones
100
25
50
75
0
fUSE5-ZZ-26.10 IgG
fUSE5-ZZ-26.10 IgG
% binding to antigen% binding to antigen
50
75
100
0
25
fUSE5-ZZ-M18 IgG
fUSE5-ZZ-M18 IgG
fUSE5-ZZ-M18 IgG
+
hIgG competitor
fUSE5-ZZ-26.10 IgG
+
hIgG competitor
2
2.5
PA
anti-M13
1
1.5

2
A
450 nm
0
0.5
Phage concentration (CFU·mL
–1
)
1.5
2
2.5
fUSE5-ZZ-M18 IgG
fUSE5-ZZ-26.10
0.5
1
1.5
A
450 nm
0
1E+07 3E+07 1E+08 3E+08 1E+09 3E+09 1E+10 3E+10
Library Cycle 1 Cycle 2 Cycle 3
A
B
C
D
Fig. 2. Characterization of fUSE5–ZZ–IgG phage. Binding analysis
of fUSE5–ZZ–IgG phage in ELISA was tested with plates coated
with PA (A) or digoxin ⁄ BSA (B). For analysis of the stability of the
ZZ–IgG complex on phage particles displaying either M18 or 26.16
mAb, fUSE5–ZZ–IgG was incubated with 1 l

M standard human IgG
as a competitor before being applied to the ELISA plates. (C) Deter-
mination of IgG molecules per phage. fUSE5–ZZ–M18 IgG phage
particles were serially diluted and applied to ELISA plates coated
with either anti-M13 IgG or PA. The number of IgG molecules per
phage particle was determined by the phage concentration derived
from PA ⁄ anti-M13 IgG in the linear range of the ELISA signals. (D)
Enrichment of fUSE5–ZZ–M18 IgG from a 1 000 000-fold excess of
fUSE5–ZZ–26.10 IgG. Phage biopanning against PA was performed
as described in Experimental procedures. Evaluation of the enrich-
ment following each round of phage selection was monitored by
phage ELISA on plates coated with PA or digoxin ⁄ BSA. ELISA
plates were developed with HRP-conjugated anti-M13 IgG; values
at 450 nm represent three independent experiments.
Selection of IgG by tandem phage panning-FACS Y. Mazor et al.
2294 FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS
falling into the double-positive quadrant were sorted
(Fig. 3). For better selectivity and for the isolation of
clones exhibiting improved dissociation rates, cells col-
lected after the first sort were immediately resorted on
the flow cytometer. As no additional probe was pro-
vided, only clones exhibiting low dissociation rates sur-
vive the second sorting cycle. IgG genes in the sort
mixture were rescued by PCR amplification, recloned
into pMAZ360–IgG, transformed into fresh NlpA–ZZ
cells, and induced for IgG expression. Following two
rounds of FACS selection, it was clear that IgGs spe-
cific for PA had been enriched (Fig. 3), and IgG genes
from the second round of FACS selection were ligated
into the pMAZ360–IgG expression vector and trans-

formed into fresh E. coli Jude-1 cells not carrying plas-
mid pBAD33–NlpA–ZZ for expression of soluble
antibodies. Individual clones were grown in 96-well
plates and induced for expression of soluble IgG, and
PA-specific clones were identified by ELISA. Forty-
nine of 192 of the screened clones gave a PA-specific
signal. Sequence analysis of 25 clones revealed the iso-
lation of six unique clones that were subjected to addi-
tional characterization. The selected clones were
expressed and purified by protein A affinity chroma-
tography, and IgG in yields of 0.5–3 mgÆL
)1
were
obtained. Biacore analysis of binding kinetics revealed
that the affinities of the IgGs derived from the immu-
nized library ranged from the low to high nanomolar
(Table 1). The highest-affinity and lowest-affinity IgGs
were determined to have K
D
values of 1 and 440 nm,
respectively.
Enrichment of high-affinity and moderate-affinity
IgGs
To evaluate the utility of tandem phage biopanning
followed by FACS for the isolation of IgGs with dif-
ferent affinities from very large libraries, we used
fUSE5–ZZ–IgG phage displaying either M18, YMF10
or VA IgG, displaying high-affinity binding (30 pm),
moderate-affinity binding (30 nm) and no binding to
the PA antigen. The high-affinity and moderate-affinity

IgGs displayed on the fUSE5-ZZ–IgG phage were
each diluted 1 : 10
8
in 10
10
copies of VA IgG displayed
on the fUSE5-ZZ–IgG phage. The phage mixture was
subjected to one round of phage biopanning, using sol-
uble biotinylated PA antigen, and, following neutral-
ization and infection of E. coli Jude-1 ⁄ F¢ cells carrying
plasmid pBAD33–ZZ, yielded an output of 1.5 ·
10
6
cells harboring phagemid pMAZ360–IgG. The cells
were induced for the expression and display of IgG,
and subjected to three rounds of FACS following
labeling with fluorescently conjugated PA63 protein.
After the third round of FACS, plasmids encoding the
isolated IgG gene inserts were transformed into fresh
E. coli cells not carrying plasmid pBAD33–NlpA–ZZ
for expression of soluble IgG and antigen specificity
screening by ELISA. Twenty-six of 186 of the screened
clones were PA-specific binders. Sequence analysis of
the positive clones confirmed the selection of the high-
affinity M18 and the moderate-affinity YMF10. To
assess the enrichment of M18 and YMF10, phagemid
was isolated from 2.5 · 10
9
cells from the pre-sort
(post-phage biopanning) and after three rounds of

Table 1. Binding kinetics of isolated IgG determined by Biacore.
Antibody k
on
(M
)1
Æs
)1
) k
off
(s
)1
) K
D
(nM)
R17 5.3 · 10
7
5.2 · 10
)2
1
R12 3.2 · 10
6
2.6 · 10
)2
8
R10 7.9 · 10
5
2.1 · 10
)2
26
R21 4.9 · 10

5
1.5 · 10
)2
31
R15 3.7 · 10
5
4.3 · 10
)2
120
42R 3.2 · 10
4
1.4 · 10
)2
440
Alexa fluor 647 (IgG expression)
0.5% 2% 7% 29%
Library 1
st
round phage panning 1
st
round FACS 2
nd
round FACS
PA-63-FITC (antigen binding)
10
2
10
3
10
4

10
5
10
2
10
3
10
4
10
5
Q4Q3
Q2Q1
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Q4Q3
Q2Q1

10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Q4
Q3
Q2Q1
10
2
10
3
10
4
10
5
10
2
10

3
10
4
10
5
Q4
Q3
Q2Q1
Fig. 3. Library selection using sequential phage biopanning and FACS. Cells were labeled with PA–FITC and Alexa Fluor 647–anti-(human
IgG) probes, and flow cytometry was used to monitor the progress of library selection by quantifying the percentage of fluorescent cells fall-
ing into the double-positive quadrant, indicating both expression and affinity. The values in quadrant 2 refer to the percentage of double-posi-
tive cells as a proportion of cells that express IgG (total cells minus quadrant 3).
Y. Mazor et al. Selection of IgG by tandem phage panning-FACS
FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS 2295
sorting. Five nanograms of purified phagemid from
each population was subjected to PCR amplification
with gene-specific primers, and the intensities of the
PCR products were determined by DNA electrophore-
sis (Fig. 4). The densities of the respective bands indi-
cated significant enrichment of both clones. Notably,
following one round of phage biopanning, a noticeable
PCR band was identified for clone YMF10 but not for
clone M18, despite there being equal numbers of cop-
ies of each clone in the initial mixture and in spite of
the much higher affinity of M18. This further empha-
sizes that library selection based on bivalent IgG dis-
play is not dictated by intrinsic affinity.
Discussion
Recombinant antibodies are routinely displayed on
phages as scFv or Fab fragments. The scFv is a mono-

mer consisting of the V
H
and V
L
gene fragments con-
nected by a peptide linker [47]. This small protein of
25 kDa is displayed very efficiently on phages, both in
monovalent (single-copy) format when fused to the
gene-3 minor coat protein in a phagemid-based system
[31], and in multivalent (multiple-copy) format when
the scFv gene is fused to all five copies of gene-3 in a
phage-based system [37]. Monovalent display systems
are more popular, as they allow the selection of anti-
bodies of higher affinity, and because it is far easier to
create large libraries in phagemids than in phages [48].
Nevertheless, scFvs often oligomerize, both when dis-
played on phages and as soluble proteins in solution,
thus making it difficult to select stringently on the
basis of intrinsic affinity; furthermore, they can be dif-
ficult to express and purify in soluble form [36,49–51].
Fab is a heterodimer consisting of the entire light
chain (V
L
–C
L
) paired with the variable and first con-
stant domain of the heavy chain (V
H
–C
H1

) [52,53]. As
opposed to scFv, the Fab molecule with a total size of
50 kDa is displayed on phages in a monovalent format
[54,55]. However, Fab display is not suitable for anti-
gens for which high-affinity binders cannot be
obtained, either because of limitations in the library
diversity, or because of the physicochemical properties
of the target (e.g. carbohydrates). Furthermore, the
display of Fab on phages is far less efficient than that
of scFv [31,36]. To address some of the limitations
associated with scFv and Fab phage display, Lee et al.
[36] recently reported the development of a system for
the display of bivalent Fab [F(ab¢)
2
] on the gene-3 coat
protein of a single phage particle in a manner that
effectively resembles the binding behavior of natural
IgGs. This display system was successfully employed
for the isolation of specific F(ab¢)
2
from synthetic
libraries [56,57]. Bivalent display results in an avidity
effect that reduces the off-rates of phage bound to
immobilized antigen or to cell surface antigens. Yet, at
the same time, the display valency is not high enough
to influence binding to soluble antigen, and thus biva-
lent phage bind to solution-phase antigen with appar-
ent affinities close to intrinsic monovalent affinities
[36]. Consequently, bivalent display systems can aid in
the recovery of antibodies with moderate affinities, and

also in selections that require dimerization for activity.
In recent years, the significance of bivalent display for
the selection of a broader spectrum of antibodies has
led to the development of several display systems that
display dimers of scFvs or Fabs to effectively mimic
the natural IgG [36,58].
Display of IgGs in their natural conformation
expands the sequence diversity that can be encoded,
and therefore increases the functional library size for
screening [38]. For this reason, we recently developed
an E. coli-based technology for the isolation of full-
length IgGs from combinatorial libraries using FACS
[39,40]. FACS is a very powerful and reliable high-
throughput screening methodology. However, sorting
of a library greater than 10
9
clones using only FACS
is time-consuming and challenging. Commercially
available flow cytometers are capable of sorting rates
of up to 40 000 s
)1
, permitting the screening of
approximately 10
8
cellsÆh
)1
[40]. Therefore, the screen-
ing of very large naı
¨
ve ⁄ synthetic libraries comprising

more than 10
9
clones would require 1 day of continu-
ous operation of the instrument, which is clearly very
challenging. This is particularly impractical if one con-
siders that at least 10 times the initial library size
should be screened to obtain efficient coverage of the
library diversity.
To reduce the initial library to a size manageable by
FACS, we describe here a method that capitalizes on
the display and selection of full-length IgGs on fila-
Pre-sort
M18
Pre-sort
YMF10
R3
YMF10
R3
M18
Fig. 4. Enrichment of high-affinity and moderate-affinity IgGs
through FACS, determined using PCR with antibody-specific prim-
ers. The amounts of M18 (30 p
M) and YMF10 (30 nM) DNA present
on this agarose gel increase following three rounds (R3) of FACS
on the phage output (Pre-sort). This indicates FACS-dependent
enrichment of these antibodies.
Selection of IgG by tandem phage panning-FACS Y. Mazor et al.
2296 FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS
mentous phage particles. For the establishment of an
IgG phage display system, we took advantage of the

fUSE5–ZZ phage. These phage particles display the
Fc-binding ZZ protein on all five copies of the phage
p3 coat protein, and are exploited as both the helper
phage and for capturing the soluble IgG via the ZZ
protein. Even though the ZZ domain has a relatively
moderate affinity for Fc IgG in solution (10 nm) [45],
its display on all copies of p3 gives rise to multivalent
display of the ZZ domain that sufficiently diminishes
the functional dissociation of the IgG. We showed that
phage particles displaying M18 IgG were efficiently
enriched from a 1 000 000-fold excess of phage dis-
playing the control 26.10 IgG. This significant enrich-
ment of specific binders from an excess of nonbinders
validated the competency of our display system, and
also provided the fundamental basis for selection of
fUSE5–ZZ–IgG from combinatorial libraries.
We took advantage of the IgG phage display system
as a prescreening step prior to selection by FACS. The
phage system provides an elegant means for the effi-
cient downsizing of very large libraries by employing
an initial round of phage selection that specifically pre-
enriches target cells from the library. The downsized
library can subsequently be subjected to rounds of
FACS, a technique that enables very precise control of
the selection process as compared with phage display
and, importantly, enables the isolation of clones exhib-
iting high affinity and selectivity.
Using an anti-PA mouse immune IgG library with
an estimated size of 2 · 10
7

as a model library, we
showed that, following an initial round of phage selec-
tion and two tandem rounds of FACS, specific anti-
PA IgGs were isolated with affinities ranging from the
low-nanmolar to mid-nanomolar range. This spectrum
of affinities emphasizes the advantages of employing
bivalent IgG display, as it facilitates the selection of
both moderate-affinity clones (R17 and R12) with
affinities in the single-digit nanomolar range and very
rare binders with modest binding kinetics (R15 and
R42) that otherwise could probably not be detected by
monovalent display. When desired, low-affinity anti-
bodies can be further engineered for improved affini-
ties by using the selected antibody gene as the basis
for subsequent mutagenesis libraries.
To demonstrate the utility of this technology with
respect to the screening of large libraries, we demon-
strated the enrichment of both a high-affinity IgG and
a moderate-affinity IgG that recognize the protective
antigen of B. anthracis from a 1 : 10
8
dilution of con-
trol antibody. Such a dilution represents only 100 mem-
bers of each specific clone in a library of 10
10
clones,
which is near the upper limit of the diversity currently
available in synthetic libraries [17,20]. We showed that
the use of sequential IgG–phage panning followed by
FACS allowed the simultaneous selection of both anti-

bodies despite the fact that they display a 1000-fold dif-
ference in affinity and each was present at a frequency
of only 1 : 100 million in the initial population.
To conclude, the significance of the methodology
described here is illustrated by the fact that it provides
the first demonstrated approach permitting the selection
of full-length IgGs from libraries displayed on phage.
We believe that sequential selection by phage display
and then FACS enables the efficient screening of very
large IgG libraries by sufficiently oversampling to cover
diversity and by simultaneously utilizing the superior
technique of FACS for final enrichment. Furthermore,
with the bivalent IgG format, it should be possible to
both select moderate-affinity antibodies from large
naı
¨
ve ⁄ synthetic repertoires, and also to affinity mature
low-affinity binders using stringent solution-phase selec-
tions that discriminate on the basis of intrinsic affinity.
Experimental procedures
Cell lines and plasmids
Phagemid pMAZ360–IgG for production of full-length IgG
has been described previously [39]. Phage fUSE5–ZZ [44]
was kindly provided by I. Benhar (Tel Aviv University,
Israel). E. coli K91K ⁄ F¢ [59] cells were used for propaga-
tion of fUSE5–ZZ and production of fUSE5–ZZ–IgG.
E. coli JUDE-1 ⁄ F¢ cells [39] (DH10B harboring the F¢
factor derived from XL1-blue) were used for expression
and purification of soluble IgG molecules.
Production of fUSE5–ZZ phage

Escherichia coli K91K ⁄ F¢ cells carrying fUSE5–ZZ DNA
were inoculated overnight at 30 C and 250 r.p.m. in 5 mL
of 2 · YT medium supplemented with 20 lgÆmL
)1
tetra-
cycline and 50 lgÆmL
)1
kanamycin. On the following day,
the culture was diluted into 500 mL of 2 · YT medium
supplemented with 20 lgÆmL
)1
tetracycline and grown
overnight at 30 °C and 250 r.p.m. Cells were pelleted at
4500 g for 15 min at 4 °C, and the supernatant was filtered
through a 0.22 lm filter. The phages were precipitated by
addition of 20% (w ⁄ v) poly(ethylene glycol) 6000 and 2.5 m
NaCl, and this was followed by centrifugation at 8000 g for
30 min at 4 °C. The phages were suspended in sterile and
filtered NaCl ⁄ P
i
at a concentration of 10
13
colony-forming
units (CFU) ⁄ mL and stored at 4 °C. To titer the phage
stock, 10-fold serial dilutions of the phages were made in
sterile 2 · YT medium. A logarithmic E. coli K91K ⁄ F¢ cul-
ture was infected with the diluted phages, and the mixed
culture was incubated for 60 min at 37 °C without shaking
Y. Mazor et al. Selection of IgG by tandem phage panning-FACS
FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS 2297

and then for 30 min with gentle shaking at 110 r.p.m.
Infected cells were plated on 2 · YT plates supplemented
with 20 l gÆmL
)1
tetracycline and 50 lgÆmL
)1
kanamycin,
and grown overnight at 37 °C.
Preparation of fUSE5–ZZ–IgG
For preparation of the fUSE5–ZZ–IgG phage particles, cul-
tures of E. coli K91K ⁄ F¢ cells transformed with phagemid
pMAZ360–IgG were grown overnight at 30 °C and
250 r.p.m. in 5 mL of 2 · YT medium supplemented with
100 lgÆmL
)1
ampicillin, 50 lgÆmL
)1
kanamycin, and 2% glu-
cose. On the following day, the cultures were diluted 1 : 100
into 10 mL of 2 · YT medium supplemented with
100 lg ⁄ mL ampicillin and 2% glucose, and grown at 37 °C
and 250 r.p.m. until 0.6 £ A
600 nm
£ 0.8. Cultures were
infected with fUSE5–ZZ helper phage at a ratio of 1 : 20
(number of bacterial cells ⁄ number of helper phage particles,
assuming that A
600 nm
= 1.0–5 · 10
8

bacteriaÆmL
)1
). The
cultures were incubated at 37 °C for 60 min without shaking,
and then with gentle shaking at 110 r.p.m. for an additional
30 min. The infected cells were collected by centrifugation at
4000 g for 10 min at 4 °C and suspended in 40 mL of
2 · YT medium supplemented with 100 lgÆmL
)1
ampicillin
and 20 lgÆmL
)1
tetracycline, and grown overnight at 30 °C
and 250 r.p.m. On the following day, the resulting fUSE5–
ZZ–IgG phage particles were precipitated with poly(ethylene
glycol) ⁄ NaCl as described above; the supernatant was care-
fully aspirated off, and the phage particles were suspended in
sterile and filtered NaCl ⁄ P
i
at a concentration of
10
11
CFUÆmL
)1
, and kept at 4 °C.
Binding analysis of fUSE5–ZZ–IgG in ELISA
ELISA plates were coated with 5 lgÆmL
)1
PA from
B. anthracis (List Biological Labs, Campbell, CA, USA) or

5 lgÆmL
)1
digoxin ⁄ BSA [60] in NaCl ⁄ P
i
at 4 °C overnight,
and then blocked with 2% (v ⁄ v) nonfat milk in NaCl ⁄ P
i
(NaCl ⁄ P
i
-M) for 2 h at room temperature. Next, 50 lLof
10
11
CFUÆmL
)1
fUSE5–ZZ–IgG phage particles was added
in a three-fold dilution series to plates already containing
100 lL of NaCl ⁄ P
i
-M and incubated for 1 h at room tem-
perature. The plates were washed three times with NaCl ⁄ P
i
containing 0.05% (v ⁄ v) Tween-20 (NaCl ⁄ P
i
-T), and bound
phage particles were detected with HRP-conjugated goat
anti-M13 IgG (antibody against pVIII) (Amersham-Pharma-
cia Biosciences, Piscataway, NJ, USA). The ELISA was
developed using the chromogenic HRP substrate TMB+
(DAKO, Glostrup, Denmark), and color development was
terminated with 1 m H

2
SO
4
. The plates were read at 450 nm.
To determine the number of IgG molecules per phage,
ELISA plates were coated with either 5 lgÆ mL
)1
goat anti-
M13 IgG (Amersham-Pharmacia Biosciences), 5 lgÆmL
)1
chicken anti-(human IgG) Fc-specific (GeneTex, Irvine, CA,
USA), or 5 lgÆmL
)1
PA in NaCl ⁄ P
i
at 4 °C for 20 h, and
blocked with 2% NaCl ⁄ P
i
-M for 2 h at room temperature.
Then, 50 lLof10
11
CFUÆmL
)1
fUSE5–ZZ–M18 IgG phage
particles were added in a three-fold dilution series to plates
already containing 100 lL of NaCl ⁄ P
i
-M and incubated for
1 h at room temperature. The plates were washed three
times with NaCl ⁄ P

i
-T, and the ELISA was developed as
above. The A
450 nm
values were plotted against the phage
concentration and used as a standard curve. fUSE5–
ZZ–M18 IgG was tested for binding to plates coated with
anti-M13 IgG, anti-Fc IgG, and PA. The number of IgG
molecules per phage was determined by calculating the ratio
of the phage concentration derived from anti-Fc IgG ⁄ anti-
M13 IgG or from PA ⁄ anti-M13 IgG in the linear range. To
evaluate the stability of the ZZ–IgG complex, freshly pro-
duced 10
11
CFUÆmL
)1
fUSE5–ZZ–IgG in NaCl ⁄ P
i
was
incubated for 1 h at room temperature with 1 lm standard
human IgG (Jackson Immunolaboratories, West Grove, PA,
USA) as a competitor prior to being applied to the ELISA
plates. The plates were washed three times with NaCl ⁄ P
i
-T,
and bound phage particles were detected with HRP-conju-
gated goat anti-M13 IgG (Amersham-Pharmacia Bioscienc-
es). The ELISA was developed using the chromogenic HRP
substrate TMB+ (DAKO), and color development was ter-
minated with 1 m H

2
SO
4
. The plates were read at 450 nm.
Enrichment of fUSE5–ZZ–M18 IgG by phage
biopanning
Anti-PA IgG-displaying fUSE5–ZZ–M18 phage particles
were enriched from a 1 000 000-fold excess of phage display-
ing the control 26.10 IgG. A 35 mm tissue culture six-well
plate was coated overnight at 4 °C with 75 lgÆ mL
)1
PA in
NaCl ⁄ P
i
in a total volume 0.7 mL. After the excess solution
had been discarded, the wells were washed once with
NaCl ⁄ P
i
and blocked with 3 mL of NaCl ⁄ P
i
containing
0.25% (w ⁄ v) gelatin (NaCl ⁄ P
i
-G) for 2 h at room tempera-
ture. Then, the wells were washed five times with NaCl ⁄ P
i
,
incubated with a phage mixture consisting of a 1 000 000-
fold excess (10
10

fUSE5–ZZ–26.10 IgG and 10
4
fUSE5–ZZ–
M18 IgG) in a total volume of 0.7 mL, and rocked gently at
room temperature for 2 h. Unbound phage particles were
rinsed away, and the plate was washed extensively 10 times
with NaCl ⁄ P
i
-T, and then five times with NaCl⁄ P
i
. Bound
phages were eluted by the addition of 400 lL of 0.1 m HCl
adjusted to pH 2.2 with glycine and 1 mg ÆmL
)1
BSA for
10 min at room temperature with gentle agitation. The eluted
phage particles were transferred into a 1.5 mL microfuge
tube, and immediately neutralized with 75 lLof1m
Tris ⁄ HCl (pH 9). The selected phage particles were used for
reinfection of E. coli K91K ⁄ F¢ cells for subsequent rounds of
phage selection. The neutralized eluted phage particles
(0.5 mL) were mixed with 4.5 mL of 2 · YT medium and
5 mL of logarithmic K91K ⁄ F¢ cells, and infection was per-
formed as described above. The infected cells were spread on
2 · YT plates supplemented with 100 lgÆmL
)1
ampicillin,
Selection of IgG by tandem phage panning-FACS Y. Mazor et al.
2298 FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS
50 lgÆmL

)1
kanamycin, and 2% glucose, and grown over-
night at 30 °C. On the following day, the plates were scraped
and subcultured into 50 mL of 2 · YT medium supple-
mented with 100 l g ÆmL
)1
ampicillin, 50 lgÆmL
)1
kanamycin
and 2% glucose to give a starting A
600 nm
of 0.1. The culture
was grown at 37 °C and 250 r.p.m. until 0.6 £ A
600 nm
£ 0.8,
and 10 mL of the culture was used for infection with fUSE5–
ZZ, as described above. Evaluation of the enrichment in each
round of selection was performed by phage ELISA as
described above.
Library selection by phage biopanning
Electrocompetent E. coli K91K ⁄ F¢ cells were transformed
with DNA phagemid of the anti-PA mouse immune library
[39] to generate a final library of 10
7
independent transfor-
mants. Library cells carrying phagemid pMAZ360–IgG
were inoculated in 500 mL of 2 · YT medium supple-
mented with 100 lgÆmL
)1
ampicillin and 2% glucose to

give a starting A
600 nm
of 0.1, and grown at 37 °C and
250 r.p.m. until 0.6 £ A
600 nm
£ 0.8. Then, the culture
was infected with fUSE5–ZZ helper phage at a ratio of
1 : 20, as described above. The infected cells were collected
by centrifugation at 4500 g for 10 min at 4 °C, suspended
in 2000 mL of 2 · YT medium supplemented with
100 lgÆmL
)1
ampicillin and 20 lgÆmL
)1
tetracycline, and
grown overnight at 30 °C and 250 r.p.m. On the following
day, fUSE5–ZZ–IgG library phages were precipitated with
poly(ethylene glycol) ⁄ NaCl as described above; the super-
natant was carefully aspirated off, and the phages were sus-
pended in sterile and filtered NaCl ⁄ P
i
at a concentration of
10
12
CFUÆmL
)1
and kept at 4 °C.
An initial cycle of phage panning was performed in solu-
tion with biotinylated PA, using streptavidin-coated para-
magnetic beads (Invitrogen, Carlsbad, CA, USA), essentially

as previously described [61]. For negative selection, the
library phages were incubated with 150 lL of streptavidin-
coated beads for 30 min at room temperature for depletion
of nonspecific fUSE5–ZZ–IgG phage particles. The phage–
bead mixture was then applied to a magnet apparatus, and
the unbound library phage particles in the supernatant were
removed to a new tube. For positive selection, depleted
library phage particles were incubated with 500 nm biotiny-
lated PA in 1 mL of NaCl ⁄ P
i
-M for 1 h at room tempera-
ture. Then, the phage–antigen complex was incubated with
150 lL of streptavidin magnetic beads for 30 min at room
temperature, and the phage–antigen–bead complex was
applied to the magnet. The beads were washed vigorously,
and bound phage particles were eluted with 1 mL of 100 mm
triethylamine (pH 13) for 10 min while being rotated. The
eluted phage particles were separated from the beads and
immediately neutralized with 200 lLof1m Tris ⁄ HCl
(pH 7.4). Library-selected phages were used for infection of
E. coli Jude-1 ⁄ F¢ cells harboring plasmid pBAD33–NlpA–
ZZ [39] for subsequent rounds of FACS selection. Neutral-
ized eluted phage particles were mixed with 4.5 mL of
2 · YT medium and 5 mL of logarithmic Jude-1–NlpA–ZZ
cells, and infection was carried out as above. The infected
cells were plated on 2 · YT plates supplemented with
100 lgÆmL
)1
ampicillin, 30 lgÆmL
)1

chloramphenicol, and
2% glucose, and grown overnight at 30 °C.
Library selection by FACS
Selection by FACS was performed essentially as previously
described [40]. Briefly, E. coli Jude-1 ⁄ F¢ cells carrying plas-
mids pBAD33–NlpA–ZZ and pMAZ360–IgG were inocu-
lated in TB medium supplemented with 100 lgÆmL
)1
ampicillin, 30 lgÆmL
)1
chloramphenicol, and 2% glucose,
and grown at 30 °CtoanA
600 nm
of 1.0. Then, cells were
collected by centrifugation at 4500 g for 10 min at 4 °C,
induced for IgG expression by resuspension in TB medium
supplemented with 100 lgÆmL
)1
ampicillin, 30 lgÆmL
)1
chl-
oramphenicol, and 1 mm isopropyl thio-b-d-galactoside
(IPTG), and grown overnight at 25 °C and 250 r.p.m. On
the following day, cells were induced with 0.2% arabinose
for an additional 3 h at 25 °C and 250 r.p.m. for NlpA–ZZ
expression. Then, the cellular outer membrane of
A
600 nm
5.0 freshly induced library cells was permeabilized
by Tris ⁄ EDTA and lysozyme treatment. For two-color

FACS based on antigen binding (affinity) and expression of
the displayed antibody, cells (10-fold excess of phage-bio-
panning-output) were labeled with 500 nm PA63–FITC
(List Biological Labs) and 100 nm Alexa Fluor 647-conju-
gated chicken anti-(human IgG) Fc-specific (GeneTex) for
2 h at 4 °C. Highly fluorescent cells falling into the double-
positive quadrant, indicating both expression and affinity,
were sorted on a FACSAria droplet deflection flow cytome-
ter (Becton Dickinson, Franklin Lakes, NJ, USA) equipped
with both a 488 and a 633 nm laser. For better selectivity,
cells captured after the first sort were immediately resorted
on the flow cytometer, using the same collection gate as
used for the initial sort. Subsequently, a DNA fragment
corresponding to the V
L
–C
K
–V
H
sequence of the IgG gene
was amplified from DNA plasmid pMAZ360–IgG of sorted
cells using the following primers: V
L
library amplifier, 5¢-
CGGATAACAATTTCACACAGG-3¢; and V
H
library
amplifier, 5¢ -AGTTCCACGACACCGTCACCG-3¢. The
PCR product was recloned into the pMAZ360–IgG vector,
retransformed into fresh Jude-1–NlpA–ZZ cells, and grown

overnight on agar plates at 30 °C. The resulting clones were
grown, induced for expression of IgG, and subjected to an
additional round of sorting.
Screening of selected clones by ELISA
Following two rounds of FACS selection, single selected
cells were screened for antigen binding in ELISA, essen-
tially as previously described [40]. Briefly, PCR-recovered
IgG genes were ligated into the pMAZ360–IgG expression
Y. Mazor et al. Selection of IgG by tandem phage panning-FACS
FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS 2299
vector and transformed into fresh E. coli Jude-1 cells (not
carrying plasmid pBAD33–NlpA–ZZ) for expression of sol-
uble, noncaptured IgGs. Randomly selected colonies were
inoculated into round-bottomed 96-well plates containing
200 lL of LB medium supplemented with 100 lgÆmL
)1
ampicillin and 2% glucose, and grown overnight at 30 °C
and 150 r.p.m. on a shaker platform. On the following day,
the cultures were diluted 1 : 20 for inoculation on fresh
round-bottomed 96-well plates containing 200 lLofTB
medium supplemented with 100 lgÆmL
)1
ampicillin and 2%
glucose, and grown for 3 h at 30 °C and 150 r.p.m. Then,
the plates were centrifuged for 10 min at 4500 g , and pellets
were resuspended in 200 lL of TB medium supplemented
with 100 lgÆmL
)1
ampicillin and 1 mm IPTG. Cells were
induced overnight at 25 °C and 150 r.p.m. for expression of

soluble IgG antibodies. On the following day, the plates
were centrifuged, and the cells were lysed in 200 lL of 20%
BugBuster HT Protein Extraction Reagent (Novagen,
Gibbstown, NJ, USA) in NaCl ⁄ P
i
for 1 h at room tempera-
ture. The plates were centrifuged as above, and soluble cell
extracts were tested for direct binding to the PA antigen as
follows. ELISA plates were coated with 5 lgÆmL
)1
PA in
NaCl ⁄ P
i
at 4 °C overnight, and blocked with 2% NaCl ⁄ P
i
-
M for 2 h at room temperature. Then, 25 lL volumes of
the cell extracts were applied to plates containing 75 lLof
NaCl ⁄ P
i
-M, and incubated for 1 h at room temperature.
The plates were washed three times with NaCl⁄ P
i
-T, and
bound IgG was detected with HRP-conjugated goat anti-
(human IgG) (Jackson Immunolaboratories). ELISA plates
were developed as above.
Expression and purification of soluble IgGs
Expression and purification of soluble full-length IgG was
performed essentially as previously described [40]. Briefly,

E. coli Jude-1 cells carrying plasmid pMAZ360–IgG were
grown at 30 °C in 200 mL of TB medium supplemented with
100 lgÆmL
)1
ampicillin and 2% glucose until the A
600 nm
was 1.0. Subsequently, cells were collected by centrifugation
at 4500 g, for 10 min at 4 °C and IgG expression was
induced by resuspension in TB medium supplemented with
100 lgÆmL
)1
ampicillin and 1 mm IPTG; cells were then
grown for 16 h at 25 °C. Induced cultures were lysed as
above, and IgGs were purified from the soluble fraction of
total cell extracts using protein A Sepharose (Amersham
Biosciences, Sweden) chromatography with final yields of
0.2–1 mgÆL
)1
of cells. Bound antibody was eluted with 0.1 m
citric acid (pH 3) and neutralized with 1 m Tris ⁄ HCl (pH 9).
Protein-containing fractions were combined, dialyzed
against 5 L of NaCl ⁄ P
i
, sterile filtered, and stored at 4 °C.
Biacore analysis
The antigen-binding kinetics of purified IgGs were deter-
mined by surface plasmon resonance analysis, using a
Biacore 3000 (Biacore-GE Healthcare, NJ, USA) instru-
ment, essentially as previously described [39]. Both a direct
binding method and a capture method were utilized to

determine kinetic parameters. Briefly, PA63 was coupled to
a CM5 chip to an equivalent of 750 response units (RU)
by using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide ⁄
N-hydroxysuccinimide chemistry as recommended by the
manufacturer. Human transferrin (Jackson Immunolabora-
tories) was similarly coupled, and used for in-line subtrac-
tion. Various concentrations of purified IgG in NaCl ⁄ P
i
were injected in duplicate at a flow rate of 50 lLÆmin
)1
for
1 min at 25 °C, and the surface was regenerated using one
pulse of 50 mm NaOH and 1 m NaCl. The data were ana-
lyzed using biaevaluation software with appropriate sub-
traction methods, and the bivalent analyte model was used
to account for avidity effects associated with the IgG
(Biacore-GE Healthcare). For the capture method, goat
anti-(human IgG
1
) Fc-specific (Jackson Immunolaborato-
ries) was coupled to a CM5 chip to an equivalent of
10 000 RU by using 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide ⁄ N-hydroxysuccinimide chemistry as recom-
mended by the manufacturer. Various concentrations of
purified IgG in Hepes-buffered saline⁄ EP buffer (Biacore,
Pittsburgh, PA, USA) were injected at a flow rate of 5 lLÆ
min
)1
at 25 °C to achieve  100 RU of captured IgGs.
Buffer and antigen were then injected serially through

in-line flow cells at a flow rate of 50 lLÆmin
)1
(5 min of sta-
bilization, 1 min of association, and 5 min of dissociation),
and the surface was regenerated using two pulses of 100 mm
H
3
PO
4
. A three-fold dilution series of PA-63, starting at
15 nm, was analyzed in duplicate using biaevaluation soft-
ware (Biacore) with appropriate subtraction methods.
Enrichment of high-affinity and moderate-affinity
IgG by tandem phage FACS
Three fUSE5–ZZ–IgG phages displaying M18, YMF10 and
VA IgGs were produced and purified as described above.
The M18, YMF10 and VA IgGs display high affinity
(30 pm) or moderate affinity (30 nm) for B. anthacis PA,
and the VA IgG binds to an unrelated antigen, the V pro-
tein of Yersinia pestis. The high-affinity and moderate-affin-
ity IgGs displayed on fUSE5–ZZ–IgG phage particles were
each diluted 1 : 10
8
in 10
11
copies of the VA IgG displayed
on fUSE5–ZZ–IgG phage particles. Phage biopanning using
biotinylated PA antigen was performed as described above.
The library was further subjected to three rounds of FACS
screening, performed essentially as described above, using

500 nm PA63–FITC and 100 nm Alexa Fluor 647-conju-
gated chicken anti-(human IgG) Fc-specific. The top 3%,
2% and 1% of fluorescent cells were collected, respectively,
in rounds 1, 2, and 3. After each round of sorting, the
insert DNA was rescued by PCR amplification, recloned
into vector pMAZ360–IgG as described above, and trans-
formed into Jude-1 cells harboring the NlpA–ZZ plasmid
Selection of IgG by tandem phage panning-FACS Y. Mazor et al.
2300 FEBS Journal 277 (2010) 2291–2303 ª 2010 The Authors Journal compilation ª 2010 FEBS
for further enrichment. After the third round of sorting,
recovered clones were transformed into Jude-1 cells and
screened by ELISA as described above. Phagemid was iso-
lated from 2.5 · 10
9
cells from the pre-sort (post-panning)
and after each of the three rounds of sorting. Five nano-
grams of DNA phagemid from each population was sub-
jected to PCR with gene-specific primers for either M18 or
YMF10. The amount of DNA encoding M18 or YMF10
IgG was determined by DNA electrophoresis.
Acknowledgements
We thank I. Benhar (Tel-Aviv University, Israel) for
providing the fUSE5–ZZ phage. This work was sup-
ported by the Clayton Foundation for Research and
by the Texas Higher Education Board Advanced
Research program. We thank the reviewers for the
many useful comments that helped us to improve the
manuscript.
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