Biopanning of antibody-phage clones using immunoplates coated with gel slices of electrophoresis: immunogel-biopanning.

Abstract

Biopanning of a phage library using a Western blotting membrane is difficult because of high background binding. We propose a reliable biopanning method, namely, immunogel-biopanning, which is performed using immunoplates coated with a molecular species fractionated from a crude sample by native polyacrylamide gel electrophoresis (PAGE). The efficacy of this method was determined in model experiments using a human interleukin-18 (IL-18)-specific single chain Fv (scFv) phage clone.

Full text

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To establish a monoclonal antibody (mAb), a phage li-
brary does not require immunization, in contrast to cell-hy-
bridization technology, which requires immunizing mice.1,2)
In a phage library, the antigen-specificity repertoire is not
substantially limited because of the absence of biological
negative clonal selection. This feature is particularly impor-
tant for the development of antibody medicine, which often
targets self-antigens. Among the revolutionary features of
antibody-displaying phage libraries, several reports have sug-
gested that a Western blotting membrane could be blotted
with a phage library, resulting in the establishment of scFvs
against new unknown molecules.3,4)However, nonspecific
binding of phage clones to membranes has hampered and re-
stricted its availability in most cases. Furthermore, SDS-
PAGE primarily resolved the denatured proteins, implying
that it is hard to isolate the antibody clones specific to a con-
formation that is critical for biological function.
To overcome these problems, we attempted to establish an
alternative approach, immunogel-biopanning, with less non-
specific binding. The two salient features of immunogel-
biopanning are that the biopanning is performed using im-
munoplates coated with proteins eluted from sliced gels of
electrophoresis and that the sensitivity to detect the target
molecule can be highly elevated with biological amplification
by using a phage clone as a probe.
In an experimental model using an established antigen-
specific phage clone, we evaluated the feasibility and sensi-
tivity of this method employing cell lysates mixed with a
minute amount of IL-18. To achieve this, we employed a na-
tive PAGE for molecular separation and an anti-IL18 svFv-
displaying phage clone, h18-108 as a probe that recognizes
the receptor-binding-site 3 of native IL-18 but not denatured
form.5)Expecting a practical experimental condition, the
amount of cell lysate was set as the highest dose of our
preparation. We demonstrated that immunogel-biopanning
was effective when cell lysates of 6.4mg contained 50ng of
IL-18 and when the phage solution of 1011cfu contained
10cfu of target-specific phage clones. DNA sequencing of
isolated phage clones after the fourth round of biopanning di-
rectly demonstrated that over 80% was the h18-108 clone.
Immunogel-biopanning is useful for the establishment of
mAbs against new native conformations, such as prion pro-
tein and for the quantitative analysis of a given target mole-
cule.
MATERIALS AND METHODS
Antibody-Phage Clone, Cell Lysate and Cytokine
tive human IL-18-specific phage clone, h18-108 was em-
ployed.5)The soluble h18-108 scFv has 50nM of Kd. A
murine neuroblastoma cell line, N2a was provided by S.
Katamine (Nagasaki University Graduate School of Medical
Science, Nagasaki). The cell lysates were prepared as de-
scribed.6)Recombinant IL-18 was purchased from MBL
(Nagoya). The concentration of protein was determined
using the DCProtein Assay (Bio-Rad Laboratories, Hercules,
CA, U.S.A.).
Electrophoresis
Samples were not denatured by heating
and SDS. Native PAGE (12.5%) was performed using run-
ning buffer without SDS as described.5,7)Gel staining was
carried out with a Silver Staining II kit (Wako, Osaka) or
Coomassie Brilliant Blue (CBB: Nacalai Tesque, Kyoto).
Immobilization of Eluted Proteins onto Immunoplates
Gels of experimental lanes were cut into several fractions.
Each gel fraction was placed in 1.7ml-tubes (Seiko,
Fukuoka, Japan) punched with a 27G needle (TERUMO,
Tokyo, Japan) and crushed by pushing them through the pore
using the gasket of a 1ml-syringe (TERUMO). Each gel
fragment was incubated with phosphate-buffered saline
(PBS) in immunoplates (F96 Maxisorp Nunc-Immuno Plate,
Nunc, Roskilde, Denmark) at 4°C. Ten hours later, the gel
fragments were discarded, and the immunoplates were
blocked with PBS containing 5% skim milk (Becton, Dickin-
son, Sparks, MD, U.S.A.). These immunoplates were used
for immunogel-biopanning as well as the enzyme-linked im-
munosorbent assay (ELISA).
ELISA
ELISA was performed as described.5,8)Briefly, a
phage solution (8?1010cfu/40ml/well) or anti-human IL-18
mAb (100ng/40ml/well, clone # 125-2H: MBL) was added
to the protein-coated wells. The phage clone was detected
using horseradish peroxidase (HRP)-conjugated anti-M13
mAb at a dilution of 1:1000 (Amersham Biosciences, Upp-
sala). Anti-human IL-18 mAb was detected with HRP-conju-
gated anti-mouse IgG at a dilution of 1:1000 (Jackson Im-
Na-
August 20071361
Biopanning of Antibody-Phage Clones Using Immunoplates Coated with
Gel Slices of Electrophoresis: Immunogel-Biopanning
Takayuki HAMASAKI,aShunsuke UCHIDA,aTomoki YOSHIHARA,aShuhei HASHIGUCHI,aYuji ITO,aand
Kazuhisa SUGIMURA*,a,b
aDepartment of Bioengineering, Faculty of Engineering, Kagoshima University; 1–21–40 Korimoto, Kagoshima,
Kagoshima 890–0065, Japan: and bCore Research for Evolutional Science and Technology, Japan Science and Technology
Corporation; Saitama 332–0012, Japan.
Received January 17, 2007; accepted May 11, 2007
Biopanning of a phage library using a Western blotting membrane is difficult because of high background
binding. We propose a reliable biopanning method, namely, immunogel-biopanning, which is performed using
immunoplates coated with a molecular species fractionated from a crude sample by native polyacrylamide gel
electrophoresis (PAGE). The efficacy of this method was determined in model experiments using a human inter-
leukin-18 (IL-18)-specific single chain Fv (scFv) phage clone.
Key words
biopanning; antibody; phage library; native gel; ELISA
Biol. Pharm. Bull. 30(8) 1361—1364 (2007)
© 2007 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed.e-mail: kazu@be.kagoshima-u.ac.jp

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munoResearch Lab., Inc., West Grove, PA, U.S.A.). After in-
cubation with a 3,3?,5,5?-tetramethylbenzidine solution, the
absorbance was measured at 450nm.
Biopanning
Biopanning was performed as described
previously.5,9)The model phage library was prepared by com-
bining IL-18-nonbinding scFv-phage clones with a fixed
number of h18-108 scFv-phage clones The unrelated phage
clones stand for the mixture of IL-18-nonbinding scFv-phage
clones. Briefly, the protein-coated wells were incubated with
the phage solution (1?1011cfu/300ml/well). Bound phages
were eluted by 0.1 M glycine-HCl (pH 2.2) and immediately
neutralized with 1 M Tris–HCl (pH 9.1). The eluates were
used for the measurement of the titer and the amplification of
the recovered phage by infection with E. coli. TG-1 as de-
scribed.5)The amplified phages were used for the subsequent
biopanning in the identical way to the first round.
DNA Sequencing
The DNA sequence of the phages was
determined using the Dye Terminator method as described.5)
RESULTS
Outline of Immunogel-Biopanning
separated by native PAGE without the treatment of SDS and
heating. After electrophoresis, running gels are fractionated
by slicing. Each gel fragment is put into immunoplate wells,
crushed to elute the proteins, and incubated overnight to coat
the wells with the eluted proteins. Both biopanning and
ELISA are performed using immunoplates treated with the
same procedure. One of the duplicated lanes is stained with
silver or CBB so that the running pattern can be visualized.
The eluted phages are propagated as described under
“Biopanning” in Materials and Methods. This procedure is
repeated several times to amplify the sensitivity and focus
the binding specificity. To determine the feasibility of this
method, we tested the model system using a specificity-de-
fined antibody phage clone.5)
An IL-18-Specific scFv-Phage Clone, h18-108, Detects
IL-18 Coated from Native PAGE Gel
separation pattern of IL-18, IL-18 alone (100ng/lane) was
run on native PAGE. A discrete major band at fraction #3 or
Crude samples are
To determine the
4 was visible by silver staining (Fig. 2a). Under the identical
condition, cell lysates mixed with 100ng of IL-18 were re-
solved by native PAGE (Fig. 2b). Expecting a practical ex-
perimental condition, the amount of cell lysate was set as the
highest dose (6.4mg) of our preparation. The control lane
was stained with CBB, while the gels of experimental lanes
were sliced for fractionation and incubated in immunoplate
wells. Although the resolved band of IL-18 was invisible by
CBB staining, a murine anti-human IL-18 mAb, 125-2H, de-
tected IL-18 in the four separated fractions, #2, 3, 4, and 5 in
ELISA (Fig. 2b). No residual IL-18 was detected in wells
coated with other fractions. Consistent with these results, the
h18-108 scFv-phage clone showed identical ability to 125-
2H for detecting IL-18, indicating that h18-108 definitely de-
tects the trace of IL-18 under experimental conditions and
also suggesting that the content in #2 and 5 appeared to be
less than the detection level of silver staining.
Detection Sensitivity Related to the Contents of Target
Protein
Cell lysates (6.4mg) mixed with varying amounts
of IL-18 (100, 50, or 10ng) were subjected to native PAGE.
As described in Fig. 1, proteins in each gel fraction were
1362Vol. 30, No. 8
Fig. 1.
After samples (8ml/lane) were separated by PAGE, running gels were fractionated by slicing gels. Each gel fragment was put into immunoplate wells, crushed to elute the pro-
teins, and incubated overnight to coat the wells with the eluted proteins. Biopanning was performed in these wells.
Outline of the Immunogel-Biopanning
Fig. 2.
out Cell Extracts
(a) Silver staining of the gel loaded with IL-18 alone. (b) Left panel: CBB staining of
gel loaded with 8ml of cell lysates (800mg/ml) mixed with IL-18. The cell lysates were
applied at the highest amounts of the prepared lysate. Right panel: Binding activity of
125-2H mAb and h18-108 scFv-phage to wells coated with IL-18 of eluates from each
gel fraction as determined by ELISA.
Native-PAGE of Recombinant Human IL-18 Mixed with or with-

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coated to immunoplates. IL-18 coated in immunoplate wells
was quantitatively estimated with ELISA using 125-2H mAb.
When the cell lysates combined with 100ng of IL-18 were
resolved on PAGE, 2, 27, 38, or 5ng of IL-18 was detected at
the gel fractions #2, 3, 4, or 5, respectively. In the case of
50ng of IL-18, the amount of the coated IL-18 was almost
1/10 of that in the case of 100ng of IL-18. IL-18 was not de-
tected in 10ng under this procedure. Biopanning was per-
formed using these immunoplates (Fig. 3). In the first round,
the protein-coated wells were incubated with 1011cfu of un-
related phage clones containing 100cfu of the h18-108 scFv-
phage. The amplified phages from each well were panned to
the same duplicated well at subsequent rounds. The numbers
of eluted phages on each round were calculated by phage
titration. After the third round, a significant number of
phages were recovered at gel fractions #3, 4, and 5 in the
case of 100ng (a) or 50ng (b) of IL-18 but not at other gel
fractions. In the case of 10ng of IL-18, no increase in the
number of phages was attained at any gel fraction. These pat-
terns were consistent with the results shown in Fig. 2, indi-
cating that this method definitely works if the crude extracts
contain 50ng of the target protein at the beginning of native
PAGE.
Minimum Number of Target-Specific Phage Clones in a
Phage Library
The requirement of the minimum number
of target-specific phage clones was estimated for the avail-
ability of a phage library to perform immunogel-biopanning.
Cell lysates (6.4mg) mixed with 100ng of IL-18 were sub-
jected to native PAGE and coated to immunoplates as de-
scribed (Fig. 1). The phage solution was prepared by mixing
a limited number of h18-108 scFv-phage clones with 1011cfu
of unrelated phage clones and put into each protein-coated
well. In the third round of biopanning, a significant increase
in the number of recovered phages was observed at #3, 4,
and 5 gel fractions when the phage solution contained
100cfu or 10cfu of the h18-108 scFv-phage (Figs. 4a,b).
The fourth round produced significantly amplified results.
Efficacy of Immunogel-Biopanning
contains a limited number of h18-108 scFv-phage clones
with 1011cfu of unrelated phage clones. The applied protein
sample contains a minute amount of target protein in the
presence of a large amount of unrelated proteins of cell
lysates. Therefore, it is important whether or not the ampli-
fied results faithfully reflect the specific amplification to con-
firm the feasibility of this method. To verify the specificity of
this amplification, the randomly selected phage clones were
directly sequenced on these scFv genes. The results demon-
strated that over 80% were the h18-108 clone (data presented
in parentheses marked with * of Fig. 4b). This result indi-
cated the reliability of this method, immunogel-biopanning.
The salient feature is that there is no increase in binding of
phages for the rest of the gel fractions, even after the fourth
round, indicating the fine specificity for this detection. This
result indicates that non-specific binding of phages as
The phage solution
August 20071363
Fig. 3.
A cell extract mixed with varying amounts of IL-18 (a: 100ng, b: 50ng, or c: 10ng/8ml/lane) was subjected to native PAGE. Immunoplates were coated with proteins contained
in each gel fraction followed by incubation with an scFv-phage solution (1011cfu) containing 100cfu of the h18-108 scFv-phage. The numbers of eluted phages were counted by
phage titration.
Immunogel-Biopanning: Content of the Target Molecule in Crude Extracts
Fig. 4.
Cell lysates mixed with IL-18 (100ng) were subjected to native PAGE. Immunoplates were coated with each gel fraction followed by incubation with an scFv-phage solution
(1011cfu) containing varying numbers of the h18-108 scFv-phage (a: 100cfu, b: 10cfu, or c: 1cfu). The eluted phage was titrated and amplified. After the fourth round, the h18-
108 clone was identified by DNA sequencing. The number in parentheses indicates the number of h18-108 clones among all clones tested (h18-108/ total tested).
Minimum Number of Target-Specific Phage Clones in a Phage Library

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pointed out in the membrane-blotting method does not ham-
per the immunogel-biopanning method. In the case of one
cfu of the h18-108 scFv-phage in the phage solution (Fig.
4c), the enrichment was not shown even in the fourth round
of biopanning. This might be attributed to non-specific loss
due to the extreme case of 1cfu of a specific clone.
DISCUSSION
Filamentous phages attach non-specifically to a Western
blotting membrane, making it difficult to enrich antigen-spe-
cific phage antibodies from phage libraries.3,4)Liu et al. re-
ported that methanol-treatment of the polyvinylidene fluoride
(PVDF) membrane could overcome this difficulty.3)However,
there have been few reports of success in the discovery of
new protein molecules using this method.
In our hands using IL-18, Liu’s protocol gave 1 to 2?106
phage-recovery (background) after the phage-amplification
eluted from 25mm2PVDF membrane coated with or without
IL-18, even if the library contained no IL-18-specific phage
clones. Therefore, we did not perform the experiments to
compare quantitatively to Liu’s method using purified IL-18
or its mixture with unrelated proteins. The purpose of this
study was, rather, to develop the panning procedure with far
less non-specific phage-binding and determine the minimum
amount of target molecule in a sample that may result from a
total loss occurring after experimental manipulations, such as
electrophoresis, gel slicing, elution efficiency, protein coat-
ing, or sensitivity of ELISA.
To simulate the practical experimental conditions, the IL-
18 was mixed with the cell lysates and analyzed for feasibil-
ity. The cell lysates contained 6.4mg/8ml/lane which was the
highest amount of sample applied in our experiments. We
showed first that immunogel-biopanning successfully worked
when the cell lysates contained 50ng of the target molecule.
The amount of 50ng can be reduced if an experimental appa-
ratus or a manipulation method is devised. Secondly, this
method works when the phage solution contains 10cfu of
target-specific phage clones in a total of 1011cfu of an unre-
lated phage population. This frequency of a target-specific
clone is quite usual in phage libraries. As the unrelated
phages were a mixture of several clones of non-IL-18 bind-
ing phage, therefore, this result was attributed to the specific
clone selection via anti-IL-18 scFv displaying on a phage
surface but not the non-specific binding via non-scFv phage
surface molecules. This result indicated that 10 out of 1011
phages were able to detect 5ng of the target protein per well
by this procedure (Figs. 3, 4). It is noteworthy that a target
protein that is invisible by even silver staining can be accu-
rately detected by antibody-phage propagation. It is also of
interest that the amplification of the detection sensitivity is
accomplished by phage propagation, while in other methods
it is usually attained by enzymatic chemical reactions or elec-
trical manipulations.
Another salient feature of our experiment is that this
method was attained using IL-18 conformation-specific scFv
phage clones. Biopanning using Western blotting is not suit-
able for isolation of conformation-specific phage clones. It is
well known that the conformation of protein is critically re-
lated to severe pathogenesis such as in amyloid b or prion
proteins.10,11)In these cases, the antibodies specific to various
conformers are promising tools for analyzing the pathogene-
sis of protein conformation diseases and development of im-
munotherapeutic reagents. The immunogel-biopanning may
be useful for this purpose. We are isolating scFv-phage
clones specific to a distinctive conformer, Barghorn’s globu-
lomer of amyloid b1—42,12)from the mixture of various con-
formers, employing this immunogel-biopanning method [in
preparation].
Thus, immunogel-biopanning is useful for the isolation of
a monoclonal antibody specific to a rare target molecule
using crude mixtures of tissue or cell lysates. This method
may also be promising for quantitative purposes and have
much higher sensitivity if it is combined with the recently re-
ported immuno-polymerase chain reaction method.13)
Acknowledgements
formed by H. Kokuryou (EIKEN CHEMICAL CO., LTD.,
Tokyo). This research was partially supported by Core Re-
search for Evolutional Science and Technology (CREST) of
the Japan Science and Technology Corporation.
Preliminary experiments were per-
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