Protein Engineering, Design & Selection vol. 23 no. 7 pp. 567– 577, 2010
Published online May 23, 2010 doi:10.1093/protein/gzq029
Antibody library screens using detergent-solubilized
mammalian cell lysates as antigen sources
Yong Ku Cho and Eric V.Shusta 1
Department of Chemical and Biological Engineering, University of
Wisconsin-Madison, Madison, WI 53706, USA
1
To whom correspondence should be addressed.
E-mail: [email protected]
Received February 3, 2010; revised April 1, 2010;
accepted April 9, 2010
Edited by James Marks
High-throughput generation of antibodies against cellular
components is currently a challenge in proteomics, therapeutic development and other biological applications. It is
particularly challenging to raise antibodies that target
membrane proteins due to their insolubility in aqueous
solutions. To address these issues, a yeast display library
of human single-chain antibody fragments (scFvs) was
efficiently screened directly against detergent-solubilized
and biotinylated lysates of a target cell line, thereby
avoiding issues with membrane protein insolubility and
eliminating the need for heterologous expression or purification of antigens. Antibody clones that specifically bind
plasma membrane proteins or intracellular proteins were
identified, depending on the biotinylation method applied.
Antibodies against a predetermined target could also be
identified using cell lysate as an antigen source as demonstrated by selecting an scFv against the transferrin receptor (TfR). When secreted from yeast and purified, the
selected scFvs are active under physiological conditions in
the absence of detergents. In addition, this method allows
facile characterization of target antigens because it is
compatible with yeast display immunoprecipitation. We
expect that this method will prove useful for multiplex
affinity reagent generation and in targeted antibody
screens.
Keywords: antibody/cell lysate/library selection/yeast display
Introduction
Affinity reagents such as antibodies that specifically bind target
proteins are an essential tool in studying key protein properties
such as expression level, cellular localization and posttranslational modification. In addition, antibodies have become
an important class of therapeutics in the past decade, as exemplified by a number of FDA approved monoclonal antibodies
for disease treatment (Schrama et al., 2006). Therefore, major
efforts to establish a comprehensive set of well-characterized
affinity reagents are under way (Taussig et al., 2007), along
with efforts to identify those that may specifically target
disease (Schrama et al., 2006; Kurosawa et al., 2008).
For systematic generation of antibody-based affinity
reagents, high-throughput in vitro technologies (Marks et al.,
1991; Kieke et al., 1997; Daugherty et al., 1998; Sheets
et al., 1998) offer several advantages over conventional
immunization approaches, since they allow rapid identification of targeting antibodies and are not limited by the
response of the immune system (Marks et al., 1991). To date,
there have been several studies using phage display to
perform proteome-wide generation of affinity reagents (Liu
et al., 2002; Schofield et al., 2007). In these cases, proteins
or protein fragments are immobilized on a matrix after being
separated in a 2D gel (Liu et al., 2002) or after being heterologously expressed (Schofield et al., 2007). However, such
an immobilization step can cause denaturation of proteins,
and 2D gel separation can lead to aggregation of membrane
proteins (Santoni et al., 2000), which are especially important targets in therapeutic applications. Moreover, several
screening methods, applying phage (Marks et al., 1993) and
yeast (Richman et al., 2006; Wang et al., 2007) display in
whole-cell panning formats have been used for isolation of
affinity reagents. Although these techniques have been successful, they have mostly focused on screening antibodies
against cell surface exposed antigens. Therefore, for
proteome-wide identification of antibodies targeted to membrane proteins and other subcellular compartments, methods
capable of subcellular, antibody-based membrane proteomics
would be desirable.
Previously, we have demonstrated that yeast displayed
scFvs allow immunoprecipitation of target antigens from
detergent-solubilized cell lysates (Cho et al., 2009). The
so-called yeast display immunoprecipitation (YDIP) method
allows facile antigen characterization and identification, as
well as quantitative detection of scFv – antigen interactions in
detergent-containing solutions by flow cytometry (Cho et al.,
2009). These findings suggested the possibility of isolating
antibodies
from
yeast
display
libraries
using
detergent-solubilized cell lysates directly as antigen sources.
Importantly, such an approach using whole-cell lysates obviates the need for heterologous expression and/or purification
of antigens and detergent use mitigates the insolubility issues
inherent to membrane proteins. In addition, since screens
would be performed in the presence of competing species,
cross-specificity and non-specificity can be easily assessed
and avoided. Here, we demonstrate that a library of nonimmune human scFvs (Feldhaus et al., 2003) can be efficiently screened using fluorescence-activated cell sorting
(FACS) and biotinylated and detergent-solubilized whole-cell
lysates from a mammalian cell line to yield target-specific
antibodies. By manipulating biotinylation conditions, antibody screening could be performed against whole-cell proteins or focused towards plasma membrane proteins,
illustrating the capability for subcellular antibody targeting.
Moreover, additional specific selection criteria can be
imposed during the screening process since antigens captured
on the yeast cell surface can subsequently be assayed with
other secondary agents. As an example, we exploited this
unique advantage to demonstrate the capability to perform
antibody screening against a predetermined target, namely
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567
Y.K.Cho and E.V.Shusta
transferrin receptor (TfR), using whole-cell lysates as the
antigen source. Antibodies arising from all of these
proof-of-concept screens are functional under physiological
conditions when produced as soluble proteins and can be
directly applied in YDIP, for characterization of specificity,
antigen size and epitope location (intracellular/extracellular).
glycine (Fisher Scientific) at 48C to completely quench
remaining NHS groups. Lysis and centrifugation were then
identical to that described for whole-cell biotinylation. The
total protein concentration remaining in the supernatant was
determined using the BCA assay per manufacturer’s instructions (Pierce, Rockford, IL).
Screening of scFv library in cell lysates using FACS
Materials and methods
Cells, media and plasmids
Saccharomyces cerevisiae strain EBY100 (Kieke et al.,
1997) and YVH10 (Shusta et al., 1998) were used for surface
display and secretion of scFvs, respectively. Surface display
plasmid pCT201-D1.3 (VanAntwerp and Wittrup, 2000) was
used for the display of anti-hen egg lysozyme D1.3 scFv.
The non-immune human scFv library (Feldhaus et al., 2003)
was a kind gift from Dr. K. Dane Wittrup at MIT. Yeast
cells were grown in the SD-CAA medium (20.0 g/l dextrose,
6.7 g/l yeast nitrogen base, 5.0 g/l casamino acids, 10.19 g/l
Na2HPO4.7H2O, 8.56 g/l NaH2PO4.H2O) at 308C to reach an
OD600nm of approximately 1.0 and induced in the same
volume of SG-CAA medium (dextrose replaced by galactose
in SD-CAA) for 16– 18 h at 208C for scFv display. The rat
brain endothelial cell line (RBE4) was a kind gift from Dr.
Roux et al. (1994). RBE4 cells were grown at 378C in 5%
CO2, in 45% alpha minimum essential medium, 45% Ham’s
F10 medium and 10% heat inactivated fetal bovine serum
(FBS, Invitrogen, Carlsbad, CA) supplemented with 100 mg/
ml streptomycin, 100 units/ml penicillin G (Invitrogen,
Carlsbad, CA), 0.3 mg/ml geneticin (Fisher Scientific,
Pittsburgh, PA) and 1 mg/l basic fibroblast growth factor
(Roche Diagnostics, Indianapolis, IN).
Biotinylation and lysis of RBE4 cells
For biotinylation and generation of whole-cell lysates,
approximately 5 106 RBE4 cells were washed in 10 mM
phosphate-buffered saline (PBS, pH 7.4) supplemented with
1 mM CaCl2, 0.5 mM Mg2SO4 (PBSCM) and incubated with
0.5 mg/ml sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) in
PBSCM for 30 min with rocking at room temperature. After
the incubation, the biotinylation solution was removed and
cells were lysed by scraping the cells into 1 ml of PBS supplemented with a protease inhibitor cocktail (Calbiochem,
Gibbstown, NJ), 2 mM EDTA and containing one of the following detergents: 1% (w/v) Triton X-100 (TX) (Sigma,
St. Louis, MO), 1% (w/v) n-octyl-b-D-glucopyranoside (OG)
(Anatrace, Maumee, OH) and 0.5% (w/v) CHAPS (Fisher
Scientific). Since residual biotinylation reagents were not
been quenched, intracellular proteins were biotinylated after
detergent lysis. The cell lysates were then centrifuged at
18 000 g for 15 min at 48C to remove insoluble debris.
After incubating the cleared lysate for 1 h at 48C, 1 M Tris at
pH 7.6 was added to a final concentration of 10 mM to
quench the reaction prior to screening so as to prevent yeast
cell biotinylation.
For biotinylation of plasma membrane proteins, the above
procedure
was
followed
except
that
0.5 mg/ml
sulfo-NHS-LC-Biotin in PBSCM is incubated for 30 min
with rocking at 48C to prevent internalization of reagent
(Bayer and Wilchek, 1990). After the biotinylation, cells
were washed three times with PBS containing 100 mM
568
Screening against whole-cell lysates or plasma membrane
proteins. A 5 107 subset of the human non-immune scFv
library was used for screening. All of the following washing
and labeling steps were performed at 48C. For the first round
of scFv screening against cell lysates (either whole cell or
plasma membrane biotinylated), 108 yeast cells were incubated overnight with 1 ml (approximately 2 mg/ml total
lysate
protein)
of
freshly
prepared
biotinylated
detergent-solubilized cell lysates supplemented with 1 mM
biotin (Fisher Scientific). Excess free biotin was added to
avoid isolation of biotin-binding scFvs. For subsequent
rounds, the number of yeast cells and the volume of lysates
were scaled accordingly. The incubated yeast cells were
washed two times with the corresponding detergent solution
and once with PBS containing 0.1% (w/v) bovine serum
albumin (PBS-BSA). The secondary detection reagents were
alternated to avoid the isolation of scFvs against these molecules (Chao et al., 2006). For the first and third rounds, a
mouse anti-c-myc antibody (9E10, 30 mg/ml, Covance,
Berkeley, CA) followed by goat anti-mouse IgG-Alexa488
conjugate (aM488, 1:500 dilution, Invitrogen, Carlsbad, CA)
was used to detect the full-length scFv expression.
Streptavidin-phycoerythrin conjugate (SA-PE, 1:80 dilution,
Sigma) was used to detect the scFv – antigen binding. In the
second round, a polyclonal rabbit anti-c-myc antibody (1:100
dilution,
Fisher
Scientific)
and
goat
anti-rabbit
IgG-allophycocyanin conjugate (aRAPC, 1:1000 dilution,
Invitrogen, Carlsbad, CA) was used to monitor the
expression. For scFv – antigen detection in the second round,
an anti-biotin monoclonal antibody (1 mg/ml, clone BTN.4,
Labvision, Fremont, CA) followed by aM488 was used.
Yeast cells that show both scFv expression and antigen
binding were isolated using Becton Dickinson FACSVantage
SE flow cytometric sorter (University of Wisconsin
Comprehensive Cancer Center). Recovered yeast cells were
grown in SD-CAA pH 4.0 (0.1 M sodium citrate, 0.1 g/L
kanamycin) for two passages to avoid bacterial growth.
Isolated scFv sequences were analyzed according to previous methods (Wang et al., 2007). Briefly, the scFv gene
was PCR amplified from yeast colonies on SD-CAA plates
using primers PNL6 forward (50 -GTACGAGCTAAAAGTACA
GTG-30 ) and PNL6 reverse (50 -TAGATACCCATACGACGTT
C-30 ). Amplified DNA was analyzed either by restriction
digest with BstNI (New England Biolabs, Ipswich, MA) or
sequencing (University of Wisconsin-Madison Biotech
Center). To verify individual clones, each plasmid was recovered from yeast using a miniprep kit (Zymed, Carlsbad, CA)
and re-transformed into the parent yeast display strain.
Targeted antibody screening: identification of TfR-associated
scFv. To demonstrate the capability of targeted antibody
screens, a TfR-associated scFv was identified. The second
round pools of plasma membrane biotinylation screens in TX
and OG lysates were subjected to additional three rounds of
Screening antibodies directly in cell lysates
screening for TfR binding. The membrane binding pools
were first incubated with freshly prepared plasma membrane
biotinylated cell lysates overnight at 48C. After incubation,
yeast cells were washed three times with corresponding lysis
buffer and incubated with a monoclonal anti-TfR antibody
(1:100 dilution, Zymed, clone H68.4, Carlsbad, CA) for 1 h
at 48C. The cells were washed and subsequently incubated
with SA-PE and aM488. Yeast cells that show both TfR
binding and cell lysate binding were isolated using Becton
Dickinson FACSVantage SE flow cytometric sorter
(University of Wisconsin Comprehensive Cancer Center).
To verify the TfR binding of the isolated antibody clone,
the antigen that was captured on yeast cells was probed with
a mouse monoclonal anti-TfR antibody (1:100 dilution,
clone OX26, Serotec) that is different from the antibody that
was used in screening. To further assess TfR association,
YDIP was performed as described below. Eluted antigen was
probed with the anti-biotin antibody (0.5 mg/ml, clone
BTN.4, Labvision) or the anti-TfR antibody (1:500 dilution,
OX26).
Identification of extracellular epitope-binding scFvs
To identify scFvs that bind extracellular epitope, plasma
membrane proteins of RBE4 cells were biotinylated and
digested using trypsin. After plasma membrane biotinylation,
RBE4 cells were incubated with 1 mg/ml of trypsin (sequencing grade, Promega, Madison, WI) in PBS at 48C for
15 min. followed by 10 min at 378C. The digestion reaction
was stopped by adding protease inhibitor cocktail (Sigma)
and 10 mM EDTA. 0.1% BSA was also added to prevent the
loss of digested fragments. The digested fragments were
incubated with the isolated yeast clones for 2 h at 48C to
determine the binding of extracellular protein fragment with
the isolated scFvs. As above, labeled yeast cells were sequentially incubated with a mouse anti-c-myc antibody followed
by aM488 mixed with streptavidin-phycoerythrin conjugate.
The fluorescence intensities were quantified using the
FACSCalibur flow cytometer (Becton Dickinson, Franklin
Lakes, NJ).
Immunolabeling using purified scFvs
The isolated scFv sequences were subcloned into secretion
vector pRS316-GAL4-4-20 (Hackel et al., 2006) as described
previously (Wang et al., 2007). In brief, the scFv ORFs were
amplified from the display vector using PNL6 primers and
were subcloned into the secretion vector as an NheI-HindIII
fragment using standard techniques. Yeast cells transformed
with scFv secretion vector were grown in 200 ml batches of
minimal SD medium (2% dextrose, 0.67% yeast nitrogen
base) supplemented with 2X SCAA amino acid (190 mg/l
Arg, 108 mg/l Met, 52 mg/l Tyr, 290 mg/l Ile, 440 mg/l Lys,
200 mg/l Phe, 1260 mg/l Glu, 400 mg/l Asp, 480 mg/l Val,
220 mg/l Thr, 130 mg/l Gly, 20 mg/l tryptophan lacking
leucine and uracil) at 308C for 72 h. Subsequently, scFv
secretion was induced at 208C for 72 h in SG-SCAA (dextrose substituted by galactose) with 0.1% (w/v) BSA. The
secreted scFv residing in the supernatant was purified using
Ni-NTA columns (Qiagen) as described previously (Hackel
et al., 2006). Protein purity was determined by SDS-PAGE
and concentration was measured using BCA assay.
Immunocytochemistry of RBE4 cells was performed as
described previously (Wang et al., 2007) with minor
modifications. Purified scFvs were pre-dimerized with
anti-c-myc antibodies (9E10) by incubating at a molar ratio
of 2:1 (scFv:antibody) in PBSCM with 1% BSA for 1 h at
48C. The concentrations of 9E10 antibody and scFv were
70 and 140 nM, respectively. To label the permeabilized
RBE4 cells, cells were first fixed with 4% paraformaldehyde
(Fisher Scientific) for 10 min on ice and permeabilized with
0.1% TX in PBS for 10 min. For unpermeabilized cells,
labeling was done without cell fixation. The pre-dimerized
scFv were incubated with RBE4 cells for 1 h at 48C followed
by a goat anti-mouse IgG-Alexa555 conjugate (aM555,
1:500 dilution, Invitrogen). Labeled cells were post-fixed
with 4% paraformaldehyde and examined with a fluorescence
microscope (Olympus IX70) or scraped off the plate in
PBS-BSA for flow cytometry.
Affinity measurement using purified scFvs
To determine the affinity of selected scFvs, RBE4 cells were
labeled with varying concentrations of purified scFvs and the
binding signal was quantified using flow cytometry. RBE4
cells grown on a 24-well plate were fixed, permeabilized
(TX) and then incubated with purified scFvs at concentrations ranging between 10 nM to 2 mM. Incubated cells
were washed and labeled with 100 nM of 9E10 epitope tag
antibody for 30 min at 48C, followed by a goat anti-mouse
IgG-Alexa555 conjugate (aM555, 1:500 dilution) for 1 h at
48C. Labeled cells were scraped off from the plate in
PBSCM with 0.1% BSA and examined using the
FACSCalibur flow cytometer. The monomeric dissociation
constant (Kd) of each clone was determined by fitting the
titration curve with a bimolecular equilibrium binding model.
YDIP of antigens
YDIP of antigens were performed with yeast cells displaying
isolated scFv clones as previously described (Cho et al.,
2009). Briefly, 108 yeast cells were incubated with 500 ml of
biotinylated cell lysate made under the same detergent condition in which the scFv had been screened for 2 h at 48C.
After the incubation, yeast cells were washed three times in
1 ml of the corresponding detergent solution at 48C.
Antigens were eluted by resuspending yeast cells in 30 ml of
0.2 M glycine – HCl solution ( pH 2.0) for 10 min. Eluted
antigens were separated in either a non-reducing or reducing
SDS-PAGE (8% separating gel), and blotted onto a nitrocellulose membrane (BioRad, Hercules, CA). Western blotting
was performed with an anti-biotin monoclonal antibody
(0.5 mg/ml, clone BTN.4, Labvision).
Results and discussion
Antibody screening against mammalian whole-cell lysates
The antibody library screening process is shown schematically
in Fig. 1. The target cellular proteins of interest are biotinylated to enable the detection of scFv– antigen interaction.
The biotinylation step can be controlled either to biotinylate
all cellular proteins or to selectively biotinylate plasma
membrane proteins (Fig. 1, Step 1). To biotinylate all proteins,
the cells are lysed in the presence of biotinylation reagent.
In contrast, for plasma membrane focused screens, a
membrane-impermeable biotinylation reagent is used before
cell lysis to selectively biotinylate plasma membrane proteins.
569
Y.K.Cho and E.V.Shusta
Fig. 1. Overview of the antibody screening method. In Step (1), target cell proteins are biotinylated and the cells are lysed using detergents. In this process,
the biotinylation conditions can be modified to biotinylate either whole-cell lysate or plasma membrane proteins. In Step 2, a yeast display scFv library is
combined with the biotinylated cell lysate from (1) to allow scFv-target protein combinations to form. In Step 3, yeast cells with scFvs that interact with a
biotinylated protein from cell lysate are screened using FACS. SA-PE is indicated as the means to detect the scFv– antigen interaction. For scFv screening
against a predetermined target such as TfR, a monoclonal anti-TfR antibody indicated as ‘known antibody’ can be used to distinguish only those scFvs that
bind to the desired target.
This approach enables subcellular screening (e.g. plasma
membrane-binding antibodies) of antibodies that would be
rare in the context of whole-cell screens due to the relatively
low expression level of membrane proteins. Next, detergents
are used to disrupt and solubilize the target cell membranes,
releasing cytosolic proteins as well as solubilized membrane
proteins to create cell lysates. The solubilized cellular proteins residing in such cell lysates are then used as sources of
antigens to screen an antibody library (Fig. 1, Step 2).
Therefore, antibodies against a target cell type of interest can
be identified without purification or heterologous expression
of antigens. In addition, since antibodies are screened
directly from a background consisting of all cellular proteins,
antibodies isolated using this method can be specifically
mined to identify those that have low or no cross-reactivity
towards other non-target cellular proteins. In the final step,
yeast cells displaying antibody clones that bind to biotinylated cell lysate proteins are then screened using FACS
(Fig. 1, Step 3).
To maximize the diversity of isolated antibodies, it is
important to use a variety of detergents that have different
solubilization characteristics. In this study, we have used TX,
OG and CHAPS that are known to have different solubilization properties. TX is known to have high solubility for
proteins (Banerjee et al., 1995) but cannot solubilize lipid
rafts (Kurzchalia et al., 1995), unlike OG, which efficiently
solubilize lipid rafts (Garner et al., 2008). CHAPS is a zwitterionic detergent, which tends to prevent non-specific aggregation of proteins better than non-ionic detergents
(Hjelmeland, 1980) and has been known to solubilize certain
membrane protein receptors better than other detergents
(Stephenson and Olsen, 1982). All three of these detergents
are also non-denaturing, a property that will help preserve
high fidelity scFv– antigen interactions in the absence of
detergent.
Thus, as a proof-of-concept demonstration of the entire
lysate-based screening procedure, we used a 5 107 clone
subset of a previously described human naı̈ve scFv library
(Feldhaus et al., 2003) combined with three rounds of FACS
screening against lysates generated from an RBE4 cell line.
Although the original library diversity was 5 108, we used
570
a fraction of the available clones such that each screening
round was amenable to flow cytometry, thus allowing
exploration of each of the different screening modalities
described above, each in multiple detergents. If desired,
established magnetic bead enrichment protocols (Yeung and
Wittrup, 2002) could be performed in the first round of
screening followed by subsequent rounds of screening using
flow cytometry in order to search larger antibody repertoires.
When the scFv library was screened using whole-cell biotinylated lysates, binding antibodies were enriched in two to
three rounds of FACS using any of the three detergents
(Fig. 2A). For all isolated pools, antibody binding was
mediated by selective interactions with cell lysate proteins
since no reactivity against secondary reagents or biotin was
detected due to the alternative use of detection reagents
applied in each round of screening and the addition of excess
biotin (1 mM) in lysates (see Materials and Methods).
Notably, pools isolated from TX and OG lysates yielded
highly enriched binding populations in two rounds, while
three rounds were required for CHAPS lysate, which might
result from varying effects of each detergent on scFv –
antigen interactions and/or protein solubility in the respective
detergents. Non-exhaustive sampling of a combined 40 scFv
sequences from the three detergent screening pools revealed
11 unique scFv sequences that when analyzed on a single
clone basis by FACS showed binding activity to biotinylated
cell lysates (Fig. 2C, Table I, whole-cell binding). As
suggested by the results of the whole pool analyses, none of
the tested clones showed binding activity for secondary
detection reagents (anti-biotin antibody or SA-PE) nor did
they bind irrelevant biotinylated proteins (biotinylated BSA
or hen egg lysozyme) (data not shown), indicating selective
interactions with cell lysate components. Of these 11 clones,
6 originated from the TX screen, 3 were from the OG screen
and 2 were from the CHAPS screen. The antibody germline
gene family usage distribution for the isolated scFv clones
was similar to a previous report that screened the same
library under physiological conditions using soluble antigens
with VH6 and VkIII/ Vl2 being most frequent (Feldhaus
et al., 2003) (Table I), indicating there is no particular bias
in antibody stability or binding in the presence of detergents.
Screening antibodies directly in cell lysates
Fig. 2. Screening of scFvs against (A) biotinylated whole-cell RBE4 lysates or (B) selectively biotinylated plasma membrane proteins residing in RBE4 cell
lysates by FACS. Flow cytometric density plots illustrating scFv clone enrichment are shown after each indicated screening round with gates used for
quantitative population analysis drawn in. Yeast cells that show both scFv expression (x-axis, detected using an anti-c-myc antibody) and lysate binding
(y-axis, detected using SA-PE) were collected in each round. Density plots from TX screens are shown. The percentages of antibody-displaying yeast cells that
bind to biotinylated lysate proteins are noted in each density plot for each detergent tested. (C) Assessment of cell lysate binding of individual clones by flow
cytometry. Representative density plots of individual clones identified from either whole-cell screening (2T5, 2O1) or plasma membrane focused screening
(3mO11) are shown. An anti-hen egg lysozyme scFv D1.3 was used as a negative control. The quantitative binding data for all individual clones are quantified
in Table I.
Complementarity determining region analysis of the 11
scFvs did not reveal significant homology, indicating reasonable diversity of isolated antibodies given the non-exhaustive
sampling of the isolated pools and pilot library size of 5 107 scFv clones. Moreover, the results suggested that it is
beneficial to perform the screening in various detergents to
maximize the diversity of isolated antibodies, and likely,
target diversity as well. Finally, the sequence diversity of isolated scFvs (11 unique sequences out of 40) compared favorably with previous antibody-endothelial cell screening
studies that used biopanning with phage display (Mutuberria
et al., 2004) (17 unique sequences out of 132) or yeast
display (Wang et al., 2007) (34 of 2000).
Antibody screening focused on plasma membrane proteins
Since antibodies that bind plasma membrane proteins have
various applications in targeted drug delivery and diagnostics, we next demonstrated lysate-based screening of antibodies targeting plasma membrane proteins. To screen
antibodies that bind membrane proteins, two separate
approaches were taken. First, antibody pools isolated after
two (TX and OG) or three (CHAPS) rounds against wholecell biotinylated lysates (Fig. 2A) were subjected to two
additional rounds of FACS with selectively membranebiotinylated cell lysates (see Materials and Methods for
details). This allowed sub-fractionation of membrane proteinbinding antibodies from amongst all binding antibodies.
After two rounds of screening against membrane proteins, a
membrane protein-binding population was only found in the
OG lysate and not in TX and CHAPS lysates (data not
shown). Moreover, even in the enriched pool against OG
lysate, only one unique clone 2O1, which was also identified
in the screen against whole-cell lysates (Table I, compare
whole-cell binding to plasma membrane binding), was identified when 20 clones were sequenced. This could be a result
of the fact that the concentration of intracellular protein is
much higher than that of plasma membrane proteins (Santoni
et al., 2000), biasing the screening towards more abundant
intracellular proteins in the first few rounds of screening
using whole-cell lysates. Alternatively, it could also be
571
Y.K.Cho and E.V.Shusta
Table I. Isolated scFv clones and their properties
Detergenta
Clonesb
Human
germline
familyc
Number
of hitsd
Whole-cell
bindinge
Plasma
membrane
bindingf
Extracellular
bindingg
Triton X-100
scFvA
scFvJj
D1.3
(NC)
4420
(NC)
2T1j
2T5
2T6
2T7
2T8
2T16
3mT23
3mT25
2O1
2O7
2O9
3mO11
3tO4j
3C1
3C3
VH3
VH3
–
Vl1
–
–
–
–
–
–
–
5.3 + 0.2
35.6 + 2.2
27.1 + 2.5
5.1 + 0.3
14.1 + 1.2
17.6 + 1.3
4.5 + 0.2
–
–
–
–
–
–
–
VH6
VH6
VH6
VH6
VH6
VH6
VH1
VH1
VH6
VH4
VH3
VH4
VH3
VH6
Vl1
Vl2
VkIII
VkIII
Vl2
Vl2
Vl1
Vl6
Vl2
VkIII
Vl2
Vl6
–
VkIII
Vl2
1
1
3
1
6
1
2
1
14
1
1
3
8
5
3
26.3 + 3.4
16.8 + 3.0
28.6 + 9.2
17.7 + 1.5
11.9 + 0.5
26.5 + 3.8
–
–
30.6 + 3.8
36.3 + 2.5
31.5 + 4.0
–
–
31.9 + 2.7
66.1 + 3.0
ND
ND
ND
ND
ND
ND
137.5 + 6.2
34.7 + 3.0
23.3 + 4.4
ND
ND
129.2 + 6.1
25.9 + 3.1
ND
ND
ND
ND
ND
ND
ND
ND
184.4 + 4.3
ND
38.4 + 0.8
ND
ND
147.1 + 3.6
ND
ND
ND
Octyl-glucoside
CHAPS
Soluble scFv
bindingh
(flow) P, UP
Soluble scFv
bindingi
(microscopy) P,
UP
–
–
–
–
–
–
–
–
–
2.0
18.8, 2.7
ND, ND
–
3.4
,0.2
0.9
3.1
4.8
2.7
1.9
1.2
,0.2
3.1
1.8
–
1.9
2.8
–
75.2, ND
–
32.0, ND
ND, ND
ND, ND
53.1, 5.4
36.2, ND
47.0, ND
–
ND, ND
46.7, 6.9
–
34.8, ND
ND, ND
–
þ þ, ND
–
ND, ND
ND, ND
ND, ND
þ þ, þ
þ, ND
þ þ, ND
–
ND, ND
þ þ, þ
–
ND, ND
ND, ND
Soluble scFv
production
(mg/l)
–, not determined; ND, not detected.
Detergent in which the scFvs have been screened from.
Name of clones. Clones names that have ‘m’ indicate that the scFv was isolated from plasma membrane-specific screening. Clone 3tO4 was screened for TfR
binding.
c
Human germline family was classified according to IgBLAST classifications (http://www.ncbi.nlm.nih.gov/igblast).
d
Number of times each clone was identified in sequence analysis.
e,f,g
Whole celle, plasma membranef, extracellular bindingg were assessed by yeast display of indicated scFv clone and measured using flow cytometry. The
indicated values are in arbitrary units proportional to fluorescence. Standard deviations from three samples are also shown. Only data for those scFvs with
binding having statistically significant differences from the negative control (NC) are listed. Those with no difference are so indicated (ND).
h,i
P, permeabilized; UP, unpermeabilized. RBE4 cells were labeled with pre-dimerized soluble scFvs at saturating concentrations followed by a secondary
antibody. hThe indicated values are in arbitrary units proportional to fluorescence and represent putative differences in antigen expression level. iPlus signs
indicate qualitative staining intensities.
j
Single-domain scFv; NC, negative control.
a
b
partially a result of the reduced library size (5 107) used in
the screens.
Thus, rather than simply subfractionating for membrane
protein-binding clones after 2 and 3 rounds of whole lysate
selections, we employed a second approach where the initial
unselected antibody pool was screened using membranebiotinylated cell lysates for every round. We applied the
initial pool of 5 107 scFvs to three rounds of screening
using TX and OG cell lysates that were selectively biotinylated at the plasma membrane. After three rounds of screening, binding populations were successfully enriched in both
detergents (Fig. 2B). Evaluating 10 clones from each detergent screen, TX clones 3mT23 and 3mT25, and OG clones
2O1 and 3mO11 were identified (Fig. 2C, Table I plasma
membrane binding). As before, no secondary reagent or irrelevant biotinylated protein binding was detected for any of
the clones tested (data not shown), suggesting specific antibody interaction with a lysate component (as further verified
in the cellular immunofluorescence and YDIP sections
below).
Through the isolation of plasma membrane protein-binding
antibodies, we have demonstrated that the screening scheme
is versatile and can be modified per application. In this
example, we adjusted the biotinylation condition to narrow
down the target antigen pool to plasma membrane proteins.
In principle, the screening procedure could also be combined
572
with various cell fractionation techniques (Stasyk and Huber,
2004; Yates et al., 2005; de Araujo et al., 2008) to identify
antibodies that target proteins in specific cellular components
and organelles such as cytoplasm, nuclei, mitochondria and
microsomes. In addition, cells directly isolated from tissue
could be used as antigen sources, which would allow antibody screenings against in vivo samples.
Targeted antibody screening: identification of TfR-associated
scFv
As another example of adjusting the screening procedure to
control the nature of scFvs isolated, it is possible to raise
antibodies against a specific target residing in
detergent-solubilized cell lysates. In the antibody screening
process, antigens are captured on the yeast surface by scFvs
(Fig. 1, Step 3). Therefore, it is possible to obtain more
information regarding the captured antigen using interactions
with other molecules as the basis for another set of screening
criteria. As one example, scFvs that are associated with a
specific target of interest can be identified by incubating
antigen-captured yeast cells with a known antibody against
the target. Such an approach allows the isolation of a panel
of antibodies recognizing a given biomarker such as a tumorspecific antigen without purification or heterologous
expression of the antigens, which is often difficult for membrane proteins (Grisshammer, 2006). This can be important
Screening antibodies directly in cell lysates
in the development of antibody therapeutics since it has been
shown that antibodies that bind to different epitopes of a
single biomarker such as epidermal growth factor receptor 2
can give rise to highly divergent pharmacodynamics due to
varying mechanism of action (Baselga and Swain, 2009).
To demonstrate this capability, we have identified an scFv
capable of binding the TfR or its associated proteins. The
TfR is a membrane protein present at the plasma membrane
and known to be expressed in the RBE4 cells used here at a
relatively high level of approximately 71 000 molecules per
cell (Huwyler et al., 1999). The plasma membrane proteinbinding pools isolated after two rounds of sorting in TX and
OG lysates (Fig. 2B) were first incubated with plasma membrane biotinylated cell lysate to capture their antigens and
subsequently incubated with a monoclonal anti-TfR antibody
that recognizes the cytosolic tail of the TfR to selectively
identify TfR-associated scFvs (Fig. 1, Step 3). Using this
approach, scFvs that possess the dual characteristics of lysate
binding and anti-TfR binding were enriched. After three
additional rounds of sorting from the second round plasma
membrane restricted pool, a single clone 3tO4 capable of
pulling the TfR out of cell lysates was identified from the
OG binding pool (Fig. 3A, Table I, plasma membrane
binding). To further confirm the result, a second monoclonal
antibody that was not used in the screening procedure was
used to probe TfR captured by yeast cells displaying scFv
3tO4 (Fig. 3B). Both the anti-TfR antibody used in the
screen and the additional anti-TfR used for the confirmation
indicated the presence of captured TfR on the surface of
yeast displaying scFv 3tO4. An irrelevant anti-hen egg lysozyme scFv D1.3 and scFv 3mO11, which binds to a non-TfR
plasma membrane protein were used as negative controls to
further verify the specificity of 3tO4 (data not shown). Thus,
it is possible to isolate an scFv (3tO4) against a specific
target of interest (TfR) by taking advantage of the yeast
display format’s capability to capture antigen. Since this particular scFv was not identified in the cursory sequencing of
10 clones as described in the previous section, the diversity
of the plasma membrane screens was likely quite good.
However, given only a single TfR-associated scFv clone was
identified, scaling up to a full antibody library size and
applying the anti-TfR screening criterion from the beginning
as opposed to the second round from a generalized plasma
membrane screening would likely expand the diversity.
Discriminating epitope localization for plasma membrane
targeting scFvs
In principle, the isolated plasma membrane protein-binding
antibodies can bind to any part of their target proteins
including extracellular or intracellular epitopes. Although
antibodies that bind to intracellular epitopes have value such
as in the generation of intrabodies (Marasco et al., 1993) or
cellular/tissue localization studies, antibodies that bind to
extracellular epitopes can have additional utility as targeted
therapeutics. Therefore, we developed an experimental
scheme to rapidly identify which of the isolated plasma
membrane antibodies targeted an extracellular epitope
(Fig. 4). Here, the plasma membrane proteins are biotinylated and the extracellular portions of biotinylated proteins
are subjected to trypsin digestion, releasing them into the
solution (Fig. 4, Step 1). The trypsin digestion is quenched
using protease inhibitors and the solution-phase extracellular
fragments of plasma membrane proteins are used to assess
whether or not the isolated scFvs recognize extracellular
Fig. 3. Identification of TfR-associated scFv. (A) Screening results after each round of FACS. The second round plasma membrane binding pool shown in
Fig. 2B was subject to an additional three rounds of screening using an anti-TfR antibody as a secondary screening criterion. X-axis indicates anti-TfR binding
(anti-TfR clone H68.4 recognizing a cytosolic epitope (White et al., 1992)) and y-axis indicates cell lysate binding (SA-PE). Gates used for quantitative
population analysis are drawn in. Single clones from the double positive population were analyzed after round 3. (B) Assessment of TfR association of scFv
3tO4. Biotinylated antigen was captured from TX solubilized lysates onto 3tO4 displaying yeast and probed with negative control antibody against rat
glyceraldehyde 3-phosphate dehydrogenase (left panel), the H68.4 antibody used in the screening (center panel), or OX-26, another anti-TfR antibody
recognizing a different epitope (extracellular) (Friden et al., 1991) (right panel). Note shift to the right of the lysate-binding population upon incubation with
anti-TfR antibodies indicating the capture of TfR on the yeast surface.
573
Y.K.Cho and E.V.Shusta
Fig. 4. Schematic of strategy for classifying scFvs capable of binding extracellular epitopes using tryptic fragments derived from intact cells. In Step 1,
biotinylated plasma membrane proteins are digested with trypsin to release extracellular fragments. The cleaved fragments are incubated with an isolated scFv
displaying yeast in Step 2. In Step 3, the interaction between extracellular fragment and scFv is quantified using a flow cytometer. If binding is retained using
the tryptic fragments, an scFv is classified as binding to an extracellular epitope (Table I).
epitopes as defined by interaction with tryptic fragments
(Fig. 4, Step 2).
To first validate this approach, two scFvs previously
selected for extracellular binding to RBE4 cells using a yeast
biopanning method (Wang et al., 2007) (scFvA and scFvJ)
were evaluated for their binding activity against tryptic
plasma membrane fragments generated from RBE4 cells.
Both scFvs bound to tryptic isolates at levels similar to that
found with membrane-biotinylated cell lysates, while an irrelevant scFv did not show any binding (Table I, compare
plasma membrane binding to extracellular binding). Among
the four membrane protein-binding clones isolated in this
study, 2O1, 3mO11 and 3mT23 showed binding activity
against trypsin-digested fragments, indicating that they bind
to extracellular epitopes (Table I), while no binding was
detected for 3mT25. Since trypsin cleaves the peptide bond
after lysine or arginine, it is possible that the antibody
epitope becomes disrupted due to the digestion, thereby generating a false-negative reading in determining extracellular
binding. In addition, it is possible that a conformational
epitope is lost upon tryptic digest or the extracellular domain
is inaccessible for enzymatic digestion as in multipass transmembrane proteins such as GPCRs. Therefore, negative
results such as those for 3mT25 in this facile test do not preclude the possibility that an scFv binds an extracellular
epitope. However, the power of the approach is that it can
allow rapid mining of scFv pools directly on the surface of
yeast for putative extracellular binding antibodies that
warrant further exploration and validation as secreted scFv
proteins. In addition, if desired more site-specific enzymes
such as Lys-C or Asp-N could be used to reduce the epitope
disruption, reducing the potential for false negatives. Yeast
display compatibility with tryptic fragments also suggests the
possibility for using protease-cleaved protein fragments
directly in antibody screens focused on targeting extracellular
domains of plasma membrane proteins.
Antibody activities under physiological conditions
Since the scFvs were screened in the presence of detergents,
it is important to confirm that the isolated scFvs bind to their
574
target under physiological conditions. Although the detergents used in the screens are known to be non-denaturing
(Hjelmeland and Chrambach, 1984), it may still be possible
that the detergent could affect the antibody or antigen conformation so that the interaction is abolished in the absence
of detergent. Moreover, although it is known that antibody –
antigen interaction is preserved in non-denaturing detergent
solutions (Cho et al., 2009; Dimitriadis, 1979), the reverse
question of whether antibodies isolated in detergent solutions
would retain their binding activity in the absence of detergents has not been well studied. Therefore, we produced all
of the full-length scFvs (excluding those that are singledomain, represented by ‘j’ in Table I) as soluble proteins and
tested their capacity to bind to the target cell line in the
absence of detergents.
Of the 13 scFvs, 11 were secreted at significant levels of
0.7– 3 mg/l while 2 were below 0.2 mg/l (Table I). These
expression levels are commensurate with what is expected
from antibody libraries in general and this library in particular (Miller et al., 2005). Binding activity was assessed by
immunolabeling RBE4 cells under physiological conditions
followed by flow cytometry or fluorescent microscopy
(Table I and Fig. 5). Significant binding was detected for 7
of the 11 well-secreted scFvs using flow cytometry and/or
fluorescence microscopy (Table I and Fig. 5). This frequency
was expected given that not all surface displayed scFvs
retain their binding activity when produced as a soluble
protein (Miller et al., 2005) and it may be possible that a
small subset of scFvs are only active in the presence of
detergents. To further characterize the binding of scFvs, the
binding affinity was determined for three of the plasma
membrane-binding clones 2O1, 3mO11 and 3mT23 by titrating soluble scFvs and quantifying the RBE4 cell binding
using flow cytometry. The dissociation constants (Kd) were
34.8 + 2.8 nM for 3mT23, 181 + 28 nM for 3mO11 and
1200 + 204 nM for 2O1 (Supplementary Fig. S1), which are
within the expected range given results using the same
library against purified soluble proteins (Feldhaus et al.,
2003) and against the same target cells using a biopanning
approach (Wang et al., 2007). When the labeling patterns of
Screening antibodies directly in cell lysates
Fig. 5. Immunolabeling of RBE4 cells with purified scFvs. Immunofluorescence patterns generated by scFvs 2T5, 2O1, 3mO11 are shown as examples. ScFv
labeling is indicated in green along with the corresponding DAPI nuclear staining in red. P denotes permeabilized cells and UP denotes unpermeabilized cells.
An anti-fluorescein scFv was used as a negative control.
scFvs were analyzed under fluorescent microscope, distinct
localization patterns such as nuclear (2T5) or punctate cell
surface (2O1) were found. Importantly, scFvs 3mO11 and
3mT23 labeled unpermeabilized cells while 3mT25 labels
only permeabilized cells (Fig. 5), corroborating the extracellular binding results using trypsin fragments (Table I).
However, for scFv 2O1 we detected immunolabeling only in
permeabilized cells (Fig. 5) while extracellular trypsin fragment labeling was detected using flow cytometry. For an
scFv like 2O1 that binds small amounts of protein in discrete
structures (Fig. 5), this discrepancy could be explained by
the fact that in flow cytometry analysis, yeast cells that
display scFv at relatively high levels (approximately 50 000
per yeast cell) are used to concentrate and detect antigens
that may be expressed at undetectable levels at the extracellular surface of the target cell using conventional immunochemistry approaches with purified scFv.
Characterization of target antigens by YDIP
Since the distinct localization patterns of scFvs suggest
differences in their target antigens, the target antigens were
characterized using YDIP (Cho et al., 2009). In YDIP, yeast
cells displaying a given scFv clone are used as affinity
reagents to immunoprecipitate the target antigen from cell
lysates. For the screens described above (whole cell, plasma
membrane and TfR focused), the entire scFv screening
process essentially follows the YDIP process, except that an
antibody library instead of a single scFv clone is used to
immunoprecipitate the target antigen. Therefore, once isolated, the antibody clones can be directly applied in YDIP
format without further optimization of target antigen solubilization or scFv – antigen interaction conditions.
YDIP antigen characterization efforts were largely focused
on clones identified from plasma membrane and TfR
screens, since these clones were of particular interest in
terms of their applicability, and also showed strong immunolabeling (Fig. 5). Thus, using YDIP, antigens for clones 2T6,
3mT23, 3mT25, 2O1, 3mO11 and 3tO4 were immunoprecipitated and subsequently detected by anti-biotin western blotting (Fig. 6). For each of these scFv clones, biotinylated
proteins were immunoprecipitated from cell lysate. As an
indication of the specificity that can be realized for isolated
scFvs, single antigen bands were immunoprecipitated for
2O1, 3mT23 and 3mT25 from a complex cell lysate.
Although the bands from 2O1, 3mO11 and 3mT23 have
similar molecular weights (Fig. 6A), bands from 2O1 and
3mO11 could be detected only after YDIP from OG lysate,
while the band from 3mT23 was detected in both TX and
OG lysates (Fig. 6A and data not shown). This suggests that
clone 3mT23 has a different antigen from clones 2O1 and
3mO11, which themselves have differing antigen localization
patterns in cellular immunofluorescence assays (Fig. 5). For
scFv 2T6, the antigen band could be only seen under reducing conditions (Fig. 6A), indicating that the antigen or
antigen complex has a large molecular weight.
In addition, we were able to further confirm the association
of 3tO4 and TfR using YDIP. The immunoprecipitated
antigen of 3tO4 was isolated and probed with both antibiotin and anti-TfR antibodies (Fig. 6B). TfR was indeed
immunoprecipitated (Fig. 6B, lane 6), although a single,
slower migrating band was present on the anti-biotin western
blot (Fig. 6B, lane 3). This result suggests that 3tO4 either
directly binds to non-biotinylated TfR that is in turn associated with another plasma membrane protein that is biotinylated (via a protein complex) or that 3tO4 binds to the
associated protein that in turn pulls down the nonbiotinylated TfR. Interestingly, TfR is biotinylated using the
protocols employed here (Wang et al., 2007), so 3tO4 is
selective for the non-biotinylated isoform. Since protein –
protein complexes are preserved under the detergent
575
Y.K.Cho and E.V.Shusta
Fig. 6. YDIP coupled with western blotting for antigen characterization. (A) Target antigens of scFvs from both whole-cell and plasma membrane screens
were immunoprecipitated from biotinylated whole-cell RBE4 lysate and probed with a monoclonal anti-biotin antibody. Lanes 1 –6 are under non-reducing
(NR) conditions and lanes 7– 12 are under reducing (R) conditions. scFvs used for YDIP were 2T6 (lanes 2 and 8), 2O1 (lanes 3 and 9), 3mO11 (lanes 4 and
10), 3mT23 (lanes 5 and 11) and 3mT25 (lanes 6 and 12). An anti-hen egg lysozyme scFv was used as a negative control (lanes 1 and 7). (B) The target
antigen of 3tO4 was purified from plasma membrane biotinylated RBE4 cell lysate and probed with anti-biotin (lanes 1– 3) or anti-TfR (lanes 4– 6) antibodies.
Lanes 1 and 4: raw plasma membrane biotinylated RBE4 cell lysate, lanes 2 and 5: YDIP from negative control scFv, lanes 3 and 6: YDIP from 3tO4. Top
arrow indicates TfR isoform immunoprecipitated by 3tO4, bottom arrow indicates the size of the biotinylated protein pulled down by 3tO4. The lower band in
lane 4 is an isoform recognized by multiple anti-TfR antibodies, but it is not pulled down by 3tO4 (not found in lane 6) and hence is not the biotinylated band
seen in lane 3.
conditions used here and many antigens function in protein
complexes, targeted TfR-type screening schemes do not
guarantee that every isolated antibody interacts directly with
the specific target. We therefore anticipate that in addition to
raising antibodies recognizing a specific antigen, concomitant raising of antibodies against the target protein complex
could find broad application. For example, antibodies against
components of protein complexes have been used to elucidate the function of each component in tumor cell migration
(Sugiura and Berditchevski, 1999) or to block a complex cellular process such as T-cell mediated cytolysis (Sarmiento
et al., 1980). Moreover, as demonstrated with 3tO4, by using
YDIP and western blotting, one can rapidly mine through the
antibody clones isolated against a specific target complex to
identify those with the desired attributes whether it be
binding to the complex or directly to the antigen of interest.
towards other cellular proteins and can be easily assayed for
said cross-reactivity (as exemplified in Fig. 6).
Another main advantage of this antibody screening strategy is the direct study of the cognate antigens using a
streamlined YDIP process. As demonstrated above, one can
readily determine the molecular weight of antigens for isolated scFvs by western blotting. It is also possible to identify
the amino acid sequence of the target antigens by applying
YDIP in combination with tandem mass spectrometry as
demonstrated previously (Cho et al., 2009). Therefore, this
method enables the discovery and detailed analysis of target
protein – antibody combinations using the cell proteome
directly and this versatile tool could have wide application in
the generation of affinity reagents.
Supplementary data
Supplementary data are available at PEDS online.
Conclusions
Here we have developed an antibody screening method that
allows identification of antibodies and cognate antigen pairs
in the context of the cellular environment by combining
detergent solubilized cell lysates and a yeast display human
scFv library. Since scFvs displayed on yeast cells are generally active in various detergent solutions commonly used in
mammalian cell lysis (Cho et al., 2009), and yeast cells
themselves are stable in various non-denaturing detergents
(Navarrete and Serrano, 1983), yeast display provides a
robust platform for antibody screening in detergent solutions.
We have demonstrated that specific scFvs functional under
physiological conditions can be isolated against diverse
target proteins including plasma membrane proteins. One of
the major advantages of this approach is that the target cell
can be directly used as the antigen source, eliminating the
need for antigen purification or heterologous expression,
which can be particularly difficult for membrane proteins. In
addition, since antibodies are screened from a background
that includes all cellular proteins, antibodies isolated using
this method are expected to have minimal cross-reactivity
576
Funding
This work was supported by National Institutes of Health
[NS052649 and EY018506]. Y.K.C. is a recipient of a
Genomic Sciences Training Program Fellowship funded
through the National Institutes of Health, 5T32HG002760.
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