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Plaque reduction test: an alternative method to assess specific antibody response to pIII-displayed peptide of filamentous phage M13
Journal of Immunological Methods 276 (2003) 175 – 183 www.elsevier.com/locate/jim
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
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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; Galfre` 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).
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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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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).
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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.
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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).
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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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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)]
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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.
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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
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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; Galfre` 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).
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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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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).
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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.
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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-
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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).
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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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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)]
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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.
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