Journal of Immunological Methods 332 (2008) 2 9

www.elsevier.com/locate/jim

Research paper
A rapid and efficient strategy to generate antigen-specific
human monoclonal antibody by in vitro immunization and
the phage display method
Shin-ei Matsumoto a , Makiko Yamashita b , Yoshinori Katakura a,b,, Yoshihiro Aiba b ,
Kosuke Tomimatsu b , Shigeru Kabayama c , Kiichiro Teruya a,b , Sanetaka Shirahata a,b
a
Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku,
Fukuoka 812-8581, Japan
b
Graduate School of Systems Life Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
c
Nihon Trim Co. Ltd., 1-8-34 Oyodonaka, Kita-ku, Osaka 531-0076, Japan
Received 28 August 2007; received in revised form 30 November 2007; accepted 5 December 2007
Available online 7 January 2008

Abstract

An in vitro immunization (IVI) protocol was developed for inducing antigen-specific immune responses in human
peripheral blood mononuclear cells (PBMCs). After antigen sensitization of PBMCs by IVI, B cells producing antigen-specific
antibody can be propagated within a week. Here, we attempted to establish a rapid, efficient strategy to obtain antigen-specific
antibody by the phage display method using in vitro immunized PBMCs. Heavy and light chain variable region genes were
easily amplified from these PBMCs immunized with mite extract (ME). After generating a combinatorial phage library
(1.6 105 members), 4 antigen-specific clones were selected by 5 panning rounds using biotinylated antigen and streptavidin
magnetic beads. Next, we combined variable region genes of these selected clones with human IgG constant region genes and
produced human IgG-type antibody. Direct and competitive enzyme-linked immunosorbent assays demonstrated that the mAb
1C11 clone bound specifically to ME. We thus established a rapid, efficient method to obtain antigen-specific human antibody
genes and produce human monoclonal IgG antibody using the phage antibody library generated from in vitro immunized
PBMCs.
2007 Elsevier B.V. All rights reserved.

Keywords: In vitro immunization; Human monoclonal antibody; Phage display

1. Introduction

Abbreviations: IVI, in vitro immunization; PBMCs, peripheral
blood mononuclear cells; mAb, monoclonal antibody; LLME, L-
leucyl-L-leucine methyl ester; CTB, cholera toxin B subunit; RA, rice
allergen; ME, mite extract; IL-2, interleukin 2; FG, Fish gelatin;
ELISPOT, enzyme-linked immunospot; scFv, single chain antibody
variable fragments.
Corresponding author.
E-mail address: katakura@grt.kyushu-u.ac.jp (Y. Katakura).
0022-1759/$ - see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jim.2007.12.005
Monoclonal antibodies (mAbs) are now considered
an epochal breakthrough in medicine and are suitable
reagents for the diagnosis and treatment of cancer,
allergy, infection, and other diseases. It is relatively easy
to raise mAbs in laboratory animals, but their therapeu-
tic efficacy is restricted by their antigenicity (Co and
Queen, 1991). Thus, human-derived mAbs are the most
 S. Matsumoto et al. / Journal of Immunological Methods 332 (2008) 29
ideal for clinical use. Thus far, several methods for
generating human mAbs have been reported, such as (1)
generation of antigen-specific scFv antibody by the
phage display method from a library generated from
human PBMCs (Barbas et al., 1991; Marks et al., 1991)
and (2) production of antigen-specific human mAbs by
introducing the human antibody gene into humanized
and/or transgenic mice (Fishwild et al., 1996). However,
these methods are time-consuming and laborious
(Vaughan et al., 1996). Therefore, we developed an in
vitro immunization protocol to augment the antigen-
specific immune response in human PBMCs (Ichikawa
et al., 1999). In this protocol, PBMCs are primarily
treated with L-leucyl-L-leucine methyl ester (LLME)
(Thiele and Lipsky, 1986; Borrebaeck et al., 1987) and
then sensitized with antigen in eRDF medium supple-
mented with IL-2, IL-4, and an adjuvant. Using this
protocol, B cells producing antigen-specific antibody
can be propagated within a week. We have thus far
succeeded in inducing antigen-specific B cells against
the cholera toxin B subunit (CTB) and rice allergen
(RA) using this IVI protocol (Shim et al., 2001; Xu et
al., 2004); however, it was difficult to achieve stable
immortalization of these antigen-specific B cells. There-
fore, we needed to obtain antigen-specific antibody
genes from the bulk culture of antigen-specific B cells
and to express recombinant human antibody.
In the present study, we attempted to establish a rapid
and efficient strategy to raise antigen-specific human
monoclonal antibody by the phage display method using
in vitro immunized PBMCs in order to obtain ME-
specific antibody genes.
2. Materials and methods
2.1. Antigen and reagents
Mite extract from Dermatophagoides farinae (ME)
was purchased from LSL (Tokyo, Japan). Recombinant
human interleukin 2 (IL-2) was purchased from
Genzyme (Cambridge, MA). Recombinant human IL-4
was purchased from Pepro Tech (London, UK). LLME
was purchased from Bachem (Torrance, CA). D-type
CpG ODN (5-ggTGCATCGATGCAGGGGggG-3)
and K-type CpG ODN (5-tcgagcgttctcC-3; uppercase
and lowercase letters indicate bases with phosphodiester
and phosphorothioate-modified backbones, respec-
tively) were purchased from Sigma Genosys (Hokkaido,
Japan) (Verthelyi et al., 2001). Fish gelatin (FG) was
purchased from BioFX Laboratories (Owings Mills,
MD). Rice allergen (RA) was prepared as previously
described (Matsuda et al., 1991).
3
2.2. Isolation of human peripheral blood mononuclear
cells
Human PBMCs from a healthy donor were separated
by density-gradient centrifugation using a lymphocyte
separation medium (LSM; Organon Teknika, Durham,
NC), as described previously (Yamashita et al., 2002;
Xu et al., 2004).
Experiments throughout this study were carried out
in accordance with the principles of the Declaration of
Helsinki and the regulations of the ethics committee of
the Faculty of Agriculture at Kyushu University.
2.3. In vitro immunization
Isolated PBMCs were first treated with 0.25 mM
LLME for 20 min at room temperature. After washing
with eRDF medium (Invitrogen, Carlsbad, CA), the
cells were sensitized with ME (10 gml 1 ) in the
presence of IL-2 (1 unitml 1 ), IL-4 (1 ngm 1 ), and
D-type CpG ODN (1 M) and cultured in eRDF
medium supplemented with 2-mercaptethanol (50 M)
and 10% heat-inactivated fetal bovine serum (FBS).
After 3 days of culture, K-type CpG ODN (1 M) was
added, and the cells were cultured for an additional
3 days.
2.4. Enzyme-linked immunospot assay
The frequency of anti-ME antibody secretion by B
cells was determined by an enzyme-linked immuno-
spot (ELISPOT) assay. Multiscreen HA filtration
plates (Millipore, Bedford, MA) were coated with
1 g of ME per well in 0.5 M carbonate buffer (pH
9.6) and incubated overnight at 4 C; thereafter, they
were blocked with 1% FG in PBS for 2 h at 37 C.
After washing the plates with PBS, in vitro immu-
nized-PBMCs in eRDF medium supplemented with
10% FBS were added to plates in triplicates at
1 105 cellswell 1 and cultured for 18 h in a
humidified atmosphere at 37 C and 5% CO2. After
the culture, the plates were washed with PBS contain-
ing 0.05% Tween 20 (PBST) and incubated with
diluted goat anti-human antibody conjugated with
horse radish peroxidase (IgM-HRP; Biosource, Camar-
illo, CA) for 2 h at 37 C. After washing the plates
with PBST, TrueBlue substrate solution (KPL,
Gaithersburg, MD) was added and the plates were
incubated at 37 C for 10 min. The reaction was
terminated by washing the plates with water, and the
plates were then dried in the dark. The number of spots
was counted using the ImageJ software.
4 S. Matsumoto et al. / Journal of Immunological Methods 332 (2008) 29
2.5. Generation of antigen-specific phage antibody by
phage display method
Total RNA from the in vitro immunized PBMCs was
extracted using the total RNA Extraction Kit (Sigma, St.
Louis, MO). Total RNA (1 g) was used as a template
for the cDNA synthesis reaction with M-MLV reverse
transcriptase (Promega, Madison, WI). The genes of the
immunoglobulin heavy chain variable region (VH) and
light chain variable region (VL) were amplified by
KOD-plus-DNA polymerase (TOYOBO, Osaka, Japan)
using appropriate family-specific primers (Marks et al.,
1991; Wang and Stollar, 2000). The amplification was
achieved by 25 to 35 polymerase chain reaction (PCR)
cycles (94 C for 15 s, 55 C for 30 s, and 68 C for
1 min). The amplified VH and VL genes were connected
by the DNA linker encoding (Gly4Ser)3 in the direction
of the 5-VH-linker-VL-3, and the recombinant scFv
fragment was then inserted into the phagemid vector
pCANTAB5E using a Recombinant Phage Antibody
System kit (Amersham Biosciences, Piscataway, NJ).
Recombinant pCANTAB5E was transfected into E.
coli TG1 cells. The transformed TG1 cells were grown
at 30 C in 2 yeast extract/bactotryptone (YT) medium
containing 2% glucose and 100 g of ampicillin per
milliliter. Ampicillin-resistant cells were infected with
M13KO7 helper phage and grown in a glucose-deficient
medium containing 100 g of ampicillin and 50 g of
kanamaycin per milliliter to yield a recombinant phage
that displayed the scFv-g3p fusion protein. A complete
phage particle was then released into the supernatant.
Next, we used streptavidin magnetic beads (Invitro-
gen) to select antigen-specific phage antibody. Prior to
panning, the magnetic beads were blocked with 2% FG or
BSA for 1 h. After phage antibodies were incubated with
biotinylated ME (10 gml 1 ) for 1 h at room
temperature, the streptavidin magnetic beads were
added. After 1 h of incubation, the beads and bound
phages were pulled down with the magnet. The beads and
bound phages were washed with 1 ml of PBS containing
0.1% Tween 20 (PBST) 7 times, with blocking buffer 2
times, and then once with PBS; finally, the bound phages
were eluted with 0.2 M glycine-HCl (pH 2.2) and used to
infect early log phase E. coli TG1 cells. After incubation
for 1 h at 37 C, the suspension was spread on SOBAG
plates containing 100 g of ampicillin per milliliter and
cultured overnight at 37 C. The next day, whole colonies
were scraped off and cultured as described above. The
phage-displayed library was constructed by the addition
of the helper phage and used for the next round of
selection. To evaluate the efficiency of the panning, the
number of colony-forming units (cfu) of E. coli producing
the acquired phage antibodies was measured before and
after each round of panning by transformation of log
phase E. coli TG1 cells with phage.
2.6. Evaluation of antigen specificity of the phage
antibody
The culture supernatants containing phage antibodies
were used for the enzyme-linked immunosorbent assay
(ELISA) to examine their binding affinity for ME.
Microtiter plates (Nunc, Roskilde, Denmark) were coated
with ME in carbonate buffer at a concentration of
10 gwell 1 and incubated overnight at 4 C; they were
then blocked with 1% FG in PBS for 2 h at 37 C. After
washing the plates, 100 l of the culture supernatants were
added to the wells and incubated for 2 h at 37 C. After
washing the plates with PBST, diluted HRP-conjugated
mouse anti-M13 mAb (Amersham Biosciences) was
added, and the plates were incubated for 2 h at 37 C.
After washing the plates with PBST, we added a substrate
solution containing 2,2-azino-bis(3-ethylbenzthiazoline-
6-sulfonic acid) (ABTS; Sigma) at a concentration of
0.3 mgml 1. Absorbance was measured at 405 nm using
a microtiter plate reader (WAKO, Osaka, Japan).
2.7. Production of recombinant human monoclonal IgG
in Chinese hamster ovary cells
We amplified the VH and VL genes using vectors
expressing scFv specific for ME as templates and ap-
propriate primers (Table 1). The VH and VL genes were
digested with Sfi I and Xho I, and with Asc I and Kas I,
respectively, and cloned into the pSecTag2A/IgGc-
bearing heavy chain constant region (CH) gene and
pSecTag2A/IgLc-bearing light chain constant region
(CL) gene, respectively, after they were digested with
the respective enzymes. About 5 106 cells of Chinese
hamster ovary (CHO) cells were transfected with these
expression vectors (5 g each) by using Lipofectamine
(Invitrogen) as described in the manufacturer's instruc-
tions. For monoclonal antibody expression, cells were
selected for DHFR expression and concurrently for
hygromycin resistance using 50 nM MTX and 300 g/
ml hygromycin B. Selected cells were cultured and the
supernatant was then collected and used for further
analysis.
2.8. Evaluation for ME-specific binding of recombinant
human IgG
The microtiter plates were coated with 1 g of ME
per well and incubated overnight at 4 C; thereafter,
 S. Matsumoto et al. / Journal of Immunological Methods 332 (2008) 29
Table 1
Oligonucleotide primers used for recombination to human antibody expression vector
For VH DNA
S1
HuJH-Xh
For V DNA
AscI-HuV1
AscI-HuV2
AscI-HuV3
AscI-HuV4
AscI-HuV5
AscI-HuV6
HuJ1-KasI-C
HuJ2-KasI-C
HuJ3-KasI-C
HuJ4-KasI-C
HuJ5-KasI-C
B : C, G or T; D : A, G or T; H : A, T or C; K : G or T; M : A or C; N : A, C, G or T;
R : A or G; S : C or G; V : A, C or G; W : A or T; Y : C or T.
they were blocked with 1% FG in PBS for 2 h at 37 C.
After washing the plates, recombinant human IgG was
added to the plates and incubated for 2 h at 37 C. The
plates were then washed 3 times with PBST, diluted
goat anti-human IgG-HRP (Biosource) was added, and
the plates were incubated at 37 C for 1 h. Thereafter,
the plates were washed again with PBST and the
substrate solution containing ABTS was added. The
absorbance was measured at 405 nm using a microtiter
plate reader.
Competitive ELISA was performed to assess the
binding of antibodies to ME. Microtiter plates were
coated with ME at a concentration of 10 gwell 1 and
incubated at 4 C overnight; they were then blocked with
1% ovalbumin in PBS at 37 C for 2 h. After washing the
plates, recombinant human IgG (8 gml 1) was added,
and they were incubated at 37 C for 2 h with serial
dilutions of ME, FG, or RA in the plates. Bound IgG
antibody was detected as above.
3. Results and discussion
3.1. IVI increased the number of B cells producing anti-
ME antibody
To induce anti-ME immune responses in PBMCs,
PBMCs were immunized in vitro with ME in presence
of IL-2, IL-4, and D- and K-type CpG ODN. After the
cells were cultured for 6 days, an ELISPOT assay was
performed to count the number of B cells producing
anti-ME antibody and evaluate the efficiency of IVI in
the induction of antigen-specific B cell responses.
5
5-CAACGTGAAAAAATTATTATTCGC-3
5-AACTCTCGAGACGGTGACCRKKGTYCC-3
5-AAAGGCGCGCCGMCATCCRGWTGACCCAGTCT-3
5-AAAGGCGCGCCGATRTTGTGATGACYCAGTCT-3
5-AAAGGCGCGCCGAAATWGTGWTGACRCAGTCT-3
5-AAAGGCGCGCCGACATCGTGATGACCCAGTCT-3
5-AAAGGCGCGCCGAAACGACACTCACGCAGTCT-3
5-AAAGGCGCGCCGAAATTGTGCTGACTCAGTCT-3
5-AAAGGGCGCCGCCACAGTTCGACGTTTGATTTCCACCTTGGTCCC-3
5-AAAGGGCGCCGCCACAGTTCGACGTTTGATCTCCAGCTTGGTCCC-3
5-AAAGGGCGCCGCCACAGTTCGACGTTTGATATCCACTTTGGTCCC-3
5-AAAGGGCGCCGCCACAGTTCGACGTTTGATCTCCACCTTGGTCCC-3
5-AAAGGGCGCCGCCACAGTTCGACGTTTAATCTCCAGTCGTGTCCC-3
Results showed that IVI increased the number of B
cells producing ME-specific antibody (Fig. 1).
3.2. Construction of an scFv phage library and
selection of antigen-specific phage antibody by panning
The variable region (V region) genes of the
antibodies produced by in vitro immunized PBMCs
were amplified by reverse transcriptase (RT)-PCR using
V-region family-specific primers, indicating that VH
gene expression in PBMCs immunized in vitro was
considerably enhanced as compared to non-stimulated
PBMCs (Fig. 2). Further, all V region family genes were
amplified using in vitro immunized PBMCs. Next, we
constructed an scFv phage library by combining the VH,
linker, and VL genes by PCR, cloning them into the
pCANTAB5E vector, and then infecting them with
M13KO7 helper phage as described in the Materials and
methods section. As a result, we obtained an scFv phage
display library containing 1.6 105 independent clones.
Next, we attempted to select antigen-specific phage
antibodies by panning using steptavidin-magnetic
beads (McConnell et al., 1999). The phage library
was incubated with biotinylated ME, and the phage
antibody that was bound to ME was captured on the
streptavidin-magnetic beads. After thorough washing,
the ME-bound phage antibody was amplified in E. coli
TG1. We repeated this selection and amplification
cycle 5 times and evaluated the efficiency of panning
(Table 2). As shown in the result, the number of re-
covered phages per applied phages increased with
repeated panning.
6 S. Matsumoto et al. / Journal of Immunological Methods 332 (2008) 29

Fig. 1. In vitro immunization augmented B cells secreting anti-ME antibody. LLME-treated PBMCs were cultured in eRDF medium supplemented with
10% FBS (no stimuli), or sensitized with ME and cultured in eRDF medium supplemented with 10% FBS, IL-2, IL-4, and CpG ODN (in vitro immunized).
After 6 days of culture, the PBMCs were seeded into Multiscreen HA plates coated with FG or ME. B cells secreting antigen-specific antibody were detected
as blue spots by Trueblue staining. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.3. Antigen specificity of phage antibody (Marks et al., 1991). To investigate the diversity of the
selected 20 clones, scFv DNA was amplified and
E. coli TG1 cells were infected with the enriched phage digested with Bst NI (Fig. 4). As shown in Fig. 4,
and induced to produce phage antibodies. Supernatants these 20 clones were found to consist of 7 individual
containing phage antibodies from 120 clones were clones. Among the 20 clones, 14 were found to be
screened by ELISA to select the antigen-specific phage identical and we designated this clone as 1C11. The
antibodies. These phage antibodies exhibited various clone diversity was also verified by sequence analysis;
antigen specificities, as shown in Fig. 3. We then selected this further revealed that 3 clones (1C08, 2G09, and
20 clones displaying a higher antigen specificity. 2E11) were not functional because of a frameshift
mutation or deletion. Thus, representative clones,
3.4. Clone diversity analysis including 1C11, 1F06, 1E04, and 2F07, were selected
for further characterization.
The digestion pattern of the V region gene obtained
using Bst NI is reported to vary in respective clones
Table 2
Efficiency of panning
Number of Input Output Efficiency
pannings performed (cfu) (cfu) (Output / Input)
1 1.0 1011 2.9 106 2.9 10 5
Fig. 2. Amplification of VH genes of antibody produced from the in
2 2.0 1012 1.3 106 6.7 10 7
vitro immunized PBMC. After 6 days of culture, total RNA was
3 6.0 1012 2.1 107 3.4 10 6
isolated from PBMCs sensitized with (in vitro immunized) or without
4 1.5 1013 2.9 108 2.0 10 5
(no-stimuli) ME, and cDNA was synthesized using total RNA. The VH
5 1.5 1013 2.7 108 1.9 10 5
genes were amplified by PCR using family-specific primers.
 S. Matsumoto et al. / Journal of Immunological Methods 332 (2008) 29 7

Fig. 3. Selection of antigen-specific phage antibody by ELISA. After 5 rounds of panning, 120 individual colonies were selected and infected with
M13KO7 helper phage to produce phage antibody. ME-specific phage antibody in the culture supernatant was detected by ELISA. FG was used as the
non-specific and negative-control antigen.
8 S. Matsumoto et al. / Journal of Immunological Methods 332 (2008) 29

Fig. 4. BstNI fingerprint analysis. ScFv DNA was amplified using a plasmid isolated from ME-specific clones as a template and digested with BstNI.
The digestion pattern was analyzed on a 4% agarose gel by electrophoresis.

3.5. Characterization of recombinant human IgG

antibodies

The VH and VL genes of these 4 scFv antibodies

were amplified and inserted into the respective expres-
sion vectors. CHO cells were transfected with these
vectors and cultured for several weeks. Supernatants
containing recombinant human IgG were collected and
their antigen specificity was investigated by ELISA
(Fig. 5). As shown in Fig. 5A, 1C11 retained its binding
specificity for ME, although other clones had lost their
antigen specificity. We further evaluated the binding
specificity of the mAb 1C11. This mAb was purified
from the culture supernatant of recombinant CHO cells
by using the MAb Trap Kit (GE Healthcare UK Ltd.,
Buckinghamshire, UK), and competitive ELISA was
performed (Fig. 5B). As shown in the result, the mAb
1C11 was shown to have a greater affinity for ME than
for FG or RA. In particular, the mAb 1C11 bound more
strongly to heat-denatured ME than to native ME. These
results suggest that the mAb 1C11 binds to the region of
ME that becomes accessible to the antibody by heat-
denaturation. Although it is necessary to determine the
accurate binding constant of 1C11 to ME, this is difficult Fig. 5. Antigen-specific binding of recombinant human IgG. A, Antigen-
specific binding of human-IgG type anti-ME antibody was evaluated by
because ME consists of many proteins extracted from
direct ELISA. VH and VL genes of phage antibodies (1C11, 1F06, 1E04,
whole body of mite. In a Western blot, the mAb 1C11 and 2F07) were amplified and inserted into the respective human IgG
can not clearly detect the target antigen, suggesting that expression vectors. CHO cells were transfected with these expression
1C11 can not bind to the SDS-denatured target antigen, vectors and cultured for several weeks. ELISA was used to evaluate the
but to soluble antigen. antigen specificity of these antibodies in the culture supernatants. The
The combined use of IVI and the phage display culture supernatant of CHO cells was used as a negative control (NC).
B, The binding-affinity of the human IgG-type mAb (1C11) was evalu-
method offers many advantages. Conventional method ated by competitive ELISA. Competitive ELISAwas performed to assess
requires an enormous phage display libraries made from the binding affinity of the mAb 1C11 for ME. A microtiter plate was
a large amount of blood of a great number of volunteers. coated with native ME. After blocking the plate, the purified mAb 1C11
IVI enables the efficient expansion of rare antigen- was added with serially diluted competitive antigens, including ME, heat-
specific B cells among the PBMCs from 10 ml of blood; denatured ME (dnME), FG, or RA. The bound mAb 1C11 was captured
by goat anti-human IgG-HRP and detected using a substrate solution
this reduces the time-consuming and laborious work containing ABTS. B/B0 is the ratio of the absorbance in the last step of
involved in the construction of an enormous library to ELISA in the presence of various concentrations of a competitive antigen
obtain antigen-specific V region genes by the phage to the absorbance in the absence of the competitive antigen.
 S. Matsumoto et al. / Journal of Immunological Methods 332 (2008) 29
display method. Furthermore, various antigens includ-
ing toxic antigen, peptide antigen and cell surface
antigen are believed to sensitize PBMCs by IVI. Here,
we demonstrated that the combined use of IVI and the
phage display method made it possible to enrich
antigen-specific B cells in PBMCs and obtain antigen-
specific antibody genes in three to six months.
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