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Journal of Immunological Methods 276 (2003) 175 – 183
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Recombinant Technology
Plaque reduction test: an alternative method to assess
specific antibody response to pIII-displayed peptide of
filamentous phage M13
Wen-Jen Yang a, David Shiuan b,*
b
a
Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan, ROC
Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, ROC
Received 30 July 2002; received in revised form 6 November 2002; accepted 15 January 2003
Abstract
Phage-displayed peptide systems have been used to identify the immunogenic epitopes and to develop the design of peptidebased or peptide-displaying phages themselves as vaccine candidates. To estimate the humoral immunity of phage-based
vaccine, it is necessary to evaluate the antibody response specifically directed at the displayed peptide. Enzyme-linked
immunosorbent assays (ELISAs) and Western blot analysis are commonly used for this purpose. However, using these methods,
it is not easy to distinguish the antibody response against phage coat protein or the antibody response specific to the displayed
peptide. The purified anti-Mycoplasma hyopneumoniae IgG was used to screen heptapeptides displaying on the pIII coat protein
of M13 phage. Four selected phage clones were chosen to immunize mice. In order to evaluate the specific antibody response
that is directed against heptapeptides, advantage was taken of the natural property of M13 phage to infect Escherichia coli,
which is mediated by the pIII coat protein binding with the F pili of E. coli, and plaque reduction tests were performed to assess
the specificity of antibody response. By comparing the number of plaques produced by the different phages (which are the same
except for the displayed peptides) neutralized by the antiserum, we could demonstrate that the specificity of antibody response
is directed against the peptide displayed on pIII coat protein. The results described here indicate that plaque reduction test is a
convenient and more precise method to detect the antibody against the phage-displayed peptide.
D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Phage display library; pIII fusion peptide; Filamentous phage M13; Plaque reduction test
Abbreviations: PR, plaque reduction; OD, optical density; BSA,
bovine serum albumin; SDS-PAGE, sodium dodecyl sulphatepolyacrylamide gel electrophoresis; NBT/BCIP, nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; PEG, polyethylene
glycol; pfu, plaque-forming unit; LB, Luria – Bertani; IPTG,
isopropyl h-D-thiogalactoside; Xgal, 5-bromo-4-chloro-3-indolylh-D-thiogalactoside.
* Corresponding author. Tel.: +886-3-8662500x21313; fax:
+886-3-8662485.
E-mail address: [email protected] (D. Shiuan).
1. Introduction
Phage display epitopes may serve as useful tools
for the development of effective vaccines for the
design of peptide-based vaccines or peptide-displaying phages themselves used for immunization (Benhar, 2001; Irving et al., 2001). Several research groups
showed that immunization with recombinant phages
could induce antibody immune responses against the
0022-1759/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0022-1759(03)00104-2
176
W.-J. Yang, D. Shiuan / Journal of Immunological Methods 276 (2003) 175–183
phage-displayed foreign peptide and cross-react with
the original target, indicating that phage-displayed
mimotopes could be used as candidates of a new
generation of subunit vaccines (Meola et al., 1995;
Bastien et al., 1997; Rudolf et al., 1998). Therefore, it
becomes crucial to assess whether or not the immune
response is generated against the mimotope while
using peptide-displaying phage as the vaccine candidate. However, the antibody response largely recognizes the wild-type phage coat proteins and is only
partially directed at the displayed peptide. Hence, it is
difficult to estimate if part of the immune response is
directed against the displaying mimotope. Several
methods have been developed to determine the antibody titer of immune response (Meola et al., 1995;
Galfrè et al., 1996). Synthesizing peptides containing
the mimotope sequence and verifying the antibody
response by ELISA or Western blotting analysis is the
most common strategy. Nevertheless, these methods
cannot easily distinguish the antibody response that is
specifically directed against the displayed peptide
from that against the phage coat proteins (Felici et
al., 1993; Meola et al., 1995).
Mycoplasma hyopneumoniae is the widespread
respiratory etiologic agent that causes the swine
enzootic pneumonia, a chronic nonfatal disease affecting pigs of all ages (Razin, 1985). The molecular
mechanism of pathogenesis is still unclear. Many
efforts to develop an effective and safe molecular
vaccine to this microorganism have been only partially successful (King et al., 1997; Fagan et al., 2001).
To understand the epitope information of M. hyopneumoniae, we biopanned a phage display peptide
library with purified anti-M. hyopneumoniae IgG.
After three rounds of biopanning, individual clones
were isolated and characterized by DNA sequencing.
Four selected phage clones were chosen to immunize
mice. ELISAs were performed to measure the titers of
selected phages that produced antisera. However, the
antibody titers contain not only antibody for mimotope but also for pIII and other coat proteins.
To demonstrate if the antibody response is directed
at the phage-displayed peptide, we utilized the plaque
reduction test to estimate the antibody response specific to the mimotope. We decided to use this test
because the random heptapeptides used in biopanning
were displayed fused to the first residue of N-terminus
of pIII. Infection of Escherichia coli by phage is
initiated by attachment of the N-terminal domain of
pIII to the tip of the pilus; therefore, this is the end of
the particle that enters the cell first. If antibody blocks
the domain, the phages will fail to infect the cells and
the plaques will not be present on the plate.
2. Materials and methods
2.1. Bacteria and animals
The E. coli strain ER2537 was used for M13
propagation (New England Biolabs, Beverly, MA,
USA). Inbred female 6- to 8-week-old specific pathogen-free BALB/cByJ mice were purchased from
National Laboratory Animal Breeding and Research
Center, Taipei, Taiwan. They were housed at the
Laboratory Animal Center, National Sun Yat-sen
University and were allowed free access to rodent
ration and water. The animal room was maintained at
23 –25 jC with a 12-h light – dark cycle. Animals
were allowed to stabilize for 10 days before the start
of the experiments to recover from any possible
stress-related effects on the immune system caused
by transportation and environmental change.
2.2. Antiserum IgG purification and characterization
The rabbit anti-M. hyopneumoniae hyperimmune
serum was prepared as described (Ro et al., 1994).
The purification of IgG from serum was based on a
modified protein A method (Bollag et al., 1996). A
protein A-agarose resin (Pierce, Rockford, IL, USA)
packed column was washed with 10 column volumes
of starting buffer (100 mM Tris – HCl, pH 7.5, 100
mM NaCl). The serum sample was mixed with equal
volume of starting buffer and applied to column. The
pass through solution was collected while measuring
OD280. The column was washed with 10 column
volumes of starting buffer until OD280 was reduced
to background levels. The IgG was eluted with 0.1 M
glycine– HCl (pH 2.5) and immediately neutralized
with 1 M Tris –HCl (pH 8.0) and stored at 20 jC
until use. The concentration of eluted IgG was estimated by OD280 (1 OD280 = 0.75 mg/ml). Western
blot analysis was used to further characterize the
eluted IgG against M. hyopneumoniae proteins
(Chung et al., 2000).
W.-J. Yang, D. Shiuan / Journal of Immunological Methods 276 (2003) 175–183
2.3. Biopanning the M13 phage-displayed random
heptapeptide library
A phage-displayed heptapeptide library (Ph.D.-7)
was purchased from New England Biolabs. The
library contains random heptapeptides followed by
a short spacer of Gly – Gly – Gly – Ser sequence
fused to the N-terminus of M13 phage minor coat
protein III. The library consisted of about 2.8 109
independent clones which should represent most of
the 1.28 109 possible seven-residue sequences. All
five copies of pIII contain the amino-terminal
random peptides (Noren and Noren, 2001). The
library was panned with purified IgG as described
below.
One hundred and fifty microliters of purified IgG
(100 Ag/ml in 0.1 M NaHCO3, pH 8.6) was dispensed
into the well of a 96-well microtiter plate (Maxisorp;
Nunc) and incubated overnight at 4 jC in a humidified container. The well was blocked with 250 Al of
blocking buffer (0.1 M NaHCO3, pH 8.6, 5 mg/ml
BSA) for 2 h at 4 jC. The well was washed six times
with TBST (TBS containing 0.1% Tween-20), then
2 1011 phages in 100 Al TBST were dispensed into
the well and rocked gently for 1 h at room temperature. Nonbinding phage were removed by washing
wells 10 times with TBST (0.1% Tween-20) in the
first round and in subsequent rounds of biopanning
with TBST (0.5% Tween-20). The bound phage were
eluted with 200 Al of elution buffer (0.2 M glycine–
HCl, pH 2.2, containing 1 mg/ml BSA) for 10 min at
room temperature and immediately neutralized with
30 Al of 1 M Tris – HCl, pH 8.0. Small aliquots of the
eluted phage were used for determination of the phage
titer. The remaining elute was used to infect E. coli
ER2537 for phage amplification.
After three rounds of biopanning, the DNA
sequences of 70 randomly selected phage clones
were determined by the dideoxy nucleotide chaintermination method using T7 Sequenase version 2.0
DNA sequencing kit (Amersham, Cleveland, OH,
USA). The single-stranded DNA of biopanningselected phages were sequenced with 58 pIII
primer (5V-CCAGACGTTAGTAAATG-3V) and the
amino acids sequences deduced. The heptapeptide
sequences of selected phages were grouped according
to the consensus amino acids they shared at the same
position.
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2.4. Binding specificity assay
Western blot analysis and competitive ELISA were
used to evaluate the binding specificity of selected
phage to purified IgG. In Western blot analysis, the
phage proteins were separated by SDS-PAGE (10%
polyacrylamide), transferred onto nitrocellulose membranes, and incubated with purified IgG. The color
development was visualized with the NBT/BCIP
substrate. The wild-type M13 phage without heptapeptide was used as a negative control. In competitive
ELISA, a constant amount of purified IgG (0.5 Ag)
and increasing amounts of the phage particles were
mixed and incubated at 37 jC for 1 h. The mixture
was applied to microtiter wells coated with mycoplasma and incubated at 37 jC for 2 h. The alkaline
phosphatase-conjugated goat anti-rabbit IgG was used
as secondary antibody and specific binding was
revealed by the addition of p-nitrophenyl phosphate
(PNPP). The automated ELISA reader (Dynex Technologies, Chantilly, VA, USA) was used to read the
OD at 405 nm.
2.5. Phage antigen preparation, animal immunization
and antisera preparation
The selected phage clones were amplified by
infecting an early-log-phase culture of E. coli
ER2537 and incubation at 37 jC with shaking for
4.5 h. The culture was centrifuged at 10,000 g for
20 min to remove the cells, the supernatant was
precipitated twice by adding 0.2 volume of polyethylene glycol solution (20% PEG-8000, 2.5 M
NaCl), incubating at 4 jC for at least 2 h before
being centrifuged at 15,000 g for 30 min at 4 jC.
The precipitated phage was resuspended in 1 ml of
TBS (50 mM Tris, 150 mM NaCl, pH 7.5) and stored
at 4 jC. Four purified phage clones were used to
immunize the mice by intraperitoneal (i.p.) administration. Each immunization used approximately 1012
pfu phage with an equal volume of TiterMax Gold
adjuvant (CytRx, Atlanta Norcross, GA, USA). Nonanesthetized animals were held and 100 Al of sample
was injected i.p. into each mouse. Tris-buffered
saline (TBS) was used as a control. Groups of three
mice were immunized for each sample. The preimmune serum was obtained and the first administration
given at day 0 followed by boosting with the same
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W.-J. Yang, D. Shiuan / Journal of Immunological Methods 276 (2003) 175–183
dose at weeks 3, 5, 7 and 14. Serum samples were
collected by tail bleeding 1 week after each booster.
Complement was inactivated by heating at 56 jC for
30 min.
mouse IgG conjugated with alkaline phosphatase was
used as the secondary antibody and incubated at room
temperature for 2 h. The detection was visualized by
the addition of NBT/BCIP substrate.
2.6. Antibody titer determination
2.7. Plaque reduction test
The ELISAs were used to determine the humoral
responses of phage immunization. The microtiter
plates were coated with 100 Al/well of 5 Ag/ml of
specific phage particle (e.g. phage clone 3 for antiphage 3 serum, wild-type M13 was used for sample
produced following TBS immunization) in coating
solution (35 mM sodium bicarbonate, pH 9.6) and
incubated at 4 jC overnight. After washing and
blocking, 100 Al/well of serial twofold dilutions of
test samples diluted with TBST (150 mM NaCl, 50
mM Tris –HCl, 0.05% Tween-20, pH 8.0) was incubated in the wells for 2 h at 37 jC. The wells were
then washed and incubated with 100 Al/well of 1:5000
diluted alkaline phosphatase-conjugated goat antimouse IgG (Zymed, South San Francisco, CA,
USA) at 37 jC for 1 h. After extensive washing with
TBST, specific binding was revealed by the addition
of p-nitrophenyl phosphate (PNPP). Color was
allowed to develop at 37 jC for 30 min and the
results were recorded by reading OD405. The titer of
antibody was determined by the reciprocal of maximum dilution factor of test serum that the OD405 is
above 0.2.
Competitive ELISAs were performed to evaluate
the ability of the phage-immunized mice sera to
compete with the rabbit anti-mycoplasma hyperimmune serum for the binding to the corresponding
phage clone. The procedure was similar to that
described above except that a constant amount of
purified rabbit anti-mycoplasma IgG (0.5 Ag) was
mixed with the serial diluted serum samples from
phage-immunized mice. Alkaline phosphatase-conjugated goat anti-rabbit IgG was used as the secondary
antibody to detect the rabbit antibody.
Western blot analysis was used to estimate the
humoral response of the pIII-displayed peptide. The
phage proteins were separated by 10% SDS-PAGE
and blotted onto a nitrocellulose membrane. After
washing and blocking, the nitrocellulose membrane
was cut into strips and incubated with serially diluted
antiserum at room temperature for 2 h. Goat anti-
The plaque reduction test was carried out by
mixing 10 Al constant amounts of phage (ca. 500
pfu) with 10 Al of diluted serum samples and then
incubating at 37 jC for 1 h. The phage – serum
mixture was used to infect 200 Al mid-log phase E.
coli ER2537 culture by incubation at room temperature for 5 min to allow the M13 phage to be adsorbed
by the E. coli. The culture was mixed with 3 ml of top
agarose, which was melted by microwave and kept in
a 45 jC water bath. The mixture was poured onto an
LB-IPTG-Xgal agar plate and incubated at 37 jC
overnight. The phage plaques produced on the plates
were counted. The following controls were performed: phage only (no serum added) and control
serum (TBS immunization). The reciprocal of the
highest dilution factor of antiserum that led to a
50% or 75% reduction in plaques was defined as
PR-50 or PR-75 titer in order to quantify the antibody
specifically against the displayed peptide on the pIII
protein.
3. Results
3.1. IgG purification and characterization of rabbit
anti-mycoplasma antiserum
The rabbit anti-M. hyopneumoniae hyperimmune
serum (Ro et al., 1994) was purified to eliminate the
interference of other serum components in the biopanning experiment. The serum IgG was eluted and
the concentration was determined by OD280. The
estimated concentration of purified IgG was 0.93
mg/ml. Western blot analysis against M. hyopneumoniae P10, P42, P60, P72 proteins (Chung et al., 2000)
and total proteins of this microorganism was used to
demonstrate whether the activity of the purified IgG
was lost during purification process. The results
showed that the purified IgG could still recognize
various recombinant proteins of M. hyopneumoniae
(Fig. 1).
W.-J. Yang, D. Shiuan / Journal of Immunological Methods 276 (2003) 175–183
179
are presented in Fig. 2A. The data indicate that the
recombinant pIII proteins can be recognized.
In competitive ELISA, the phage particles can
compete with purified IgG binding to the microtiter
wells coated with mycoplasma. The competition
results of two selected phage clones and wild-type
M13 are shown in Fig. 2B. The data showed that
wild-type M13 phage did not interfere with IgG
binding to mycoplasma; however, the selected phage
clones could reduce the purified IgG binding to
mycoplasma. Taken together, these data indicate that
the selected heptapeptides of phage clones could bind
specifically to the purified IgG and mimic the epitopes
on M. hyopneumoniae.
Fig. 1. Characterization of purified IgG. Western blotting analysis
was performed using purified IgG against E. coli expressing
M. hyopneumoniae proteins. Lane 1: protein marker (the molecular
weight is indicated on the left); lane 2: P10 protein; lane 3: P42
protein; lane 4: P60 protein; lane 5: P72 protein; lane 6: M. hyopneumoniae total proteins.
Table 1
Classification of the deduced amino acid sequences alignment of
selected heptapeptides obtained after three rounds of biopanninga
Group 1
3.2. Sequences analysis of selected heptapeptides
After three rounds of biopanning, individual
phage clones were isolated and characterized by
DNA sequencing. The number of pfu obtained after
each round of biopanning increased gradually, indicating that the biopanning selections were successful. The deduced amino acid sequences were
analyzed. There are 38 different sequences among
the 70 selected clones. Twenty-one of the selected
phage clones could be divided into three groups according to the shared amino acids (Table 1). Three
consensus sequences F_QDL, SI_PT(L)S(L)L and
NAPP were found among these sequences. These
motifs may be important epitopes of M. hyopneumoniae antigens.
3.3. Specific interactions between the purified IgG
and selected phages
Each selected phage was separated by SDS-PAGE
and blotted onto a nitrocellulose membrane to investigate whether the purified IgG could recognize the
fused heptapeptide displayed on the pIII protein of
M13 phages. All of the 38 different sequences were
recognized by Western blot to different degrees. Six
selected clones with the different recognition levels
Y
Y
T
T
F
F
F
F
F
F
F
N
A
S
N
T
R
–
Q
Q
Q
Q
Q
Q
Q
D
D
D
D
D
D
D
I
L
L
L
N
M
L
M
Q
H
H
G
G
P
S
S
S
S
S
S
S
L
Y
L
L
S/L
L
L
G
L
L
L
L
T
L
L
L
Q
Q
H
H
H
T
L
L
P
R
P
P
P
P
M
M
Q
Q
V
A
M
T
L
Group 2
V
I
I
I
A
S
H
I
S
V
P
A
S
H
T
T
–
P
P
L
N
N
P
P
P
P
T
T
T
K
Q
L
L
L
A
A
T/L
Group 3
I
T
T
Y
N
N
N
G
A
A
A
A
A
P
P
P
H
E
P
A
T
S
S
S
S
S
N
a
M
V
S
A
L
L
A
The selected sequences could be classified into three groups
according to the shared amino acid sequences. Sequences are shown
using the single-letter amino acid code. Amino acids that are
identical within the heptapeptides are shown in bold upper case. The
conserved amino acids found in more than three different selected
clones are summarized as bold-italic-type letters.
180
W.-J. Yang, D. Shiuan / Journal of Immunological Methods 276 (2003) 175–183
Fig. 2. Binding activity of selected phages to purified IgG. (A) Western blot analysis shows the interaction of isolated phage clones with protein
A purified rabbit anti-M. hyopneumoniae IgG. The levels of IgG recognition of six selected phage clones (sequences indicated on the right) and
wild-type M13 were shown. The position of heptapeptide fused to pIII is indicated by the arrow. No binding was found to pIII on wild-type M13
phage. M: protein marker, the molecular weights are indicated on the left. (B) Competitive between phage particles and purified IgG for binding
to coated mycoplasma. Two selected phage clones; f51: AEPVAML (square), f58: SISNLLL (triangle) and wild-type M13 phage (diamond)
were shown.
3.4. Measurements of antibody responses to selected
phage clones
ELISAs were performed to measure the serum
IgG immune responses of selected phage clones.
Serum samples 1 week after the final booster were
collected from three mice (each immunized with the
same phage) and each sample was assayed in duplicate. The antibody responses against specific phage
clones are shown in Fig. 3A. The highest antibody
titers, about 218, were obtained 1 week after final
booster. However, this assay measures antibodies
against M13 coat proteins (e.g. pIII, pVIII, etc.) as
well as those against heptapeptide displayed on pIII
protein.
The anti-peptide titers of sera were determined by
Western blotting of pIII-displayed peptides of phage
clones. Giving serum-3 as an example (Fig. 3B), the
level of recognition of pIII protein decreased with
the dilution factor of serum. The band corresponding
to pIII protein disappeared when serum-3 was
diluted in 1:215 (Fig. 3B, panel 1, lane 7). These
data show that the titer of serum-3 against pIII of f3
was 214. However, this titer includes antibody that
recognized other parts of pIII protein. The TBSimmunized serum cannot recognize the pIII protein
of f3 (panel 2).
In order to further determine the titer specific for
pIII-displayed peptide, competitive ELISAs were
performed to detect the ability of phage-immunized
mice sera to compete with the rabbit anti-mycoplasma hyperimmune serum for binding to the corresponding phage clone. As shown in Fig. 4, serum-3
did not interfere with the binding of rabbit anti-
W.-J. Yang, D. Shiuan / Journal of Immunological Methods 276 (2003) 175–183
Fig. 3. Antibody responses against specific phage clones. (A) The
serum samples were collected from mice 1 week after final booster
and ELISA used to measure each sample in duplicate. The specific
phage particles corresponding to the serum samples were used in the
assay except wild-type M13 for TBS-produced serum. The antigen
samples which produced their own sera are represented by the
following symbols: f3, SSTPALL (w); f12, AVVPKSL (5); f31,
TIANLSL (4); f58, SISNLLL (); TBS (o). (B) Western blot
analysis of the anti-pIII-displayed peptide titer of serum-3 on pIII
protein of f3 (panel 1) and TBS-immunized serum on pIII protein
of f3 (panel 2). Serum samples were serial twofold dilution from
1:29 (lane 1) to 1:215 (lane 7).
181
plaques of phage clones f3 filled the plate when
serum-3 was not added (Fig. 5A, left). However, the
phage plaques were reduced significantly when an
equal amount of phage clone f3 (as Fig. 5A, left)
was incubated with serum-3 (1:128 dilution) as
described in Section 2.7 (Fig. 5A, right). The results
indicate that plaques are not formed when antibodies block the N-terminal domain of M13 phage
pIII protein. In contrast, plaques of phage clones
f31 filled the plate when no serum was added (Fig.
5B, left). Interestingly, the plaques still spread all
over the plate while incubated with serum-3 (1:128
dilution) (Fig. 5B, right). These data show that
serum-3 could not neutralize phage clones f31.
However, the phage clones f3 and f31 contain
the same phage particle components except for the
heptapeptide displayed on the N-terminal of pIII
protein. We could therefore deduce that the antibody
responses are directed against the displayed heptapeptide.
The PR-50 titers of selected phage clone-produced
antisera in this study were estimated. Giving serum-3
as an example, as shown in Table 2, the plaque counts
of phage clone f3 without serum-3 were 787 pfu. The
plaque numbers reduced to 387 pfu when serum-3
(1:512 dilution) was added. According to the definition of PR-50 described in Section 2.7, the PR-50 titer
mycoplasma serum to phage clone 3 when diluted
2048-fold. These data indicate that the titer specific
for phage 3-displayed peptide was 1024. However,
the TBS-immunized serum did not interfere with the
binding of rabbit anti-mycoplasma serum to phage
clone 3.
3.5. Assessing the specific antibody response to pIII
fusion peptide by plaque reduction test
Plaque reduction tests were performed to further
estimate whether the antibody responses were specific against the heptapeptide displayed on pIII protein. The results of two phage clones f3 (SSTPALL)
and f31 (TIANLSL) neutralized by f3-produced
sera (named serum-3) are shown in Fig. 5. The
Fig. 4. Competitive ELISAs of the f3 or TBS-immunized mice
serum compete with the rabbit anti-mycoplasma hyperimmune
serum for the binding to f3 clone. The negative control (no serum
added) is indicated on the figure. The serum-3 and TBS-produced
serum are indicated in black and white bars, respectively. The data
are presented as means with standard deviations.
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W.-J. Yang, D. Shiuan / Journal of Immunological Methods 276 (2003) 175–183
Fig. 5. Plaque reduction tests of serum-3 against different phage clones. (A) The plaques of phage clone f3 produced on the plate without
addition of serum-3 are shown on the left. The plaques produced on the plate when an equal amount of phage clone f3 was incubated with
serum-3 (1:128 dilution) are shown on the right. (B) The plaques of phage clone f31 produced on the plate when no serum-3 was added (left).
The plaques of phage clone f31 produced on the plate when incubated with 1:128 diluted serum-3 (right).
of serum-3 was estimated as 512. The data also
indicates that TBS-produced serum could not interfere
with the plaque formation of any selected phage clone
(Table 2).
4. Discussion
The phage display system has been applied extensively in various fields (Benhar, 2001). Applications
Table 2
The plaque counts of serial diluted serum-3 against different phage clones
Phage clone
Plaque counts
Serum-3 dilution factor
1:128
f3, SSTPALL
f12, AVVPKSL
f31, TIANLSL
f58, SISNLLL
a
b
100.
12
750
752
738
(98%)b
(0.04%)
(0.03%)
(0.04%)
1:256
1:512
1:1024
1:2048
186
762
755
746
387
754
767
753
603
763
753
750
780
772
769
745
(76%)
(0.02%)
(0.03%)
(0.03%)
(51%)
(0.03%)
(0.01%)
(0.02%)
(23%)
(0.02%)
(0.03%)
(0.02%)
(0.01%)
(0.01%)
(0.01%)
(0.03%)
The dilution factor of TBS produced serum was 1:128.
The percentage of plaque reduction is indicated in parenthesis. The value was determined as [(pfuno
No serum-3
TBS seruma
787
781
776
765
784
772
770
748
(0%)
(0%)
(0%)
(0%)
(0.01%)
(0.01%)
(0.01%)
(0.02%)
serum pfuserum add)/(pfuno serum)] W.-J. Yang, D. Shiuan / Journal of Immunological Methods 276 (2003) 175–183
in new drug development, vaccine and diagnostic
agents are very promising. The molecular pathogenesis mechanism of M. hyopneumoniae remains
elusive, we do not understand which protein(s) of
this pathogen play important roles in pathogenesis.
In this study, rabbit anti-mycoplasma hyperimmune
serum was used to screen the phage-displayed peptide library. From the point of view of vaccine
development, using polyclonal antibody to screen
epitopes may obtain overall crucial epitope information of the pathogen without detailed approaches in
pathogenesis. The consensus sequences found in this
study may be located at the epitopes of proteins that
have not been published. It is also possible that these
consensus sequences correspond to the conformational epitopes. Further studies are ongoing to evaluate the potential of these selected phage clones for
developing vaccine candidates.
Using a synthetic peptide containing the mimotope
sequence to verify antibody response by ELISA or
Western blotting is a well-used strategy. Nevertheless,
it is difficult to validate the mimotope-specific antibody response. In addition, synthesizing peptides is
time-consuming, expensive, cannot be done in a
standard immunology laboratory and some sequences
may no longer be recognized by specific antibodies
when they are not displayed on phages (Felici et al.,
1993; Meola et al., 1995). The merit of using plaque
formation as the criteria to estimate specific antibody
response is that it is an available standardized technique for phage amplification and titer determination in
the laboratory. The results presented in this study
indicate that plaque reduction test is a convenient
and more precise method for assessing specific antibody response to pIII-displayed peptide of filamentous phage M13.
Acknowledgements
This research was supported in part by grant NSC
88-2311-B110-004 from the National Science Council, Republic of China. We thank Dr. A.J.T. George
and J. Sullivan for reading the manuscript and many
valuable suggestions.
183
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