Journal of Dermatological Science, 4 (1992) 26-32 0
26
DESC
1992 Elsevier
Science
Publishers
B.V. All rights
reserved.
0923-181
l/92/$05.00
00148
Production
and characterization
of monoclonal
antibodies to Treponema
pallidurn Kyu Kwang
Whang,
Min Geol
Lee ‘, Wook
Lew ’ and Jung Bock
Departments of Dermatology. Konkuk University College of Medicine, Chungkuk, Korea and
Lee ’
’ Yonsei Universitv College of Medicine,
Seoul. Korea (Received
13 December
Key words:
1990; accepted
Monoclonal
27 December
1991)
Treponema pallidurn
antibody;
Abstract This study
was attempted
to produce
the monoclonal
antibodies
for Treponema pallidum and to investigate
specific
teristics, thereby contributing to identify the antigenic structure and to apply the diagnosis YS 75, YS 307, YS 481, YS 343, YS 1 and YS 406) secreting monoclonal antibodies reactive isotypes of the seven monoclonal from 0.59 to 1.48 as measured sensitized pathogenic clonal
with
T. pallidurn, showed
treponemes,
antibodies
antibodies by ELISA.
from
ies from YS 343 reacted
but the other
strong
produced were defined. Monclonal antibodies fluorescences.
6 monoclonal
5 of the 7 clones with a polypeptide
reacted
Optical densities of the 7 monoclonal from 5 of the 7 clones which could
The monoclonal
antibodies
reacted
with a polypeptide
of 64 kDa common
antibody
to both
antibodies agglutinate
were ranged sheep RBC
from YS 343 was cross-reactive
with T. pa/lidum specifically.
of a molecular
their charac-
of syphilis. The seven clones (YS 3, with T. pallidzrm were produced. The
weight
of 47 kDa,
On immunoblotting. and monoclonal
T. pallidum and T. phagedenis. The above results
with nonmonoantibodrevealed
that 7 clones secreting monoclonal antibodies reactive to T. pallidum were produced successfully, and specific antibodies reacting with major antigenic polypeptides of a molecular weight of 47 kDa would be useful in the diagnosis of syphilis in the future.
Introduction Treponema pallidurn, T. pertenue and T. carateum inducing syphilis, yaw and pinta, respectively, are included among pathogenic treponemes; T. denticola, T. microdentium, and T. phagedenis, T. refringens, and T. vincentii, which reside normally in oral, genital, and gastroenteric mucosa, are included among nonpathoCorrespondence to: J.B. Lee, Department of Dermatology, Yonsei University College of Medicine, CPO Box 8044, Seoul, Korea.
genie treponemes [ 11. Nonpathogens can be cultured in broth thioglycollate media, but T. pallidum can be cultured only in rabbit testicle; consequently, so relatively small amounts can be obtained. For this reason, there are many restrictions in pathogenic, immunodiagnostic, and preventive studies. Therefore there have been studies of monoclonal antibodies specific for T. pallidurn and its recombinant antigenic cloning genetically [2]. But these should be preceded by biological and biochemical identifications of polypeptides of T. pallidurn. Those polypeptides were separated on sodium
21
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were electrophoretically transferred to a nitrocellulose paper for antigenic analysis. Eight to 23 protein antigens of T. pallidum are identifiable today [ 3-71. The number of common antigens with T. phagedenis were reported as 5,6, 8, 11 or 15, differing according to authors and measuring methods [ 3,6,8,9]. We have observed that immunodominant antigens were polypeptides of 47, 36.5, 29.5, 15.5 and 14 kDa, and that 11 antigens were common to both T. palhdum and T. phagedenis [ 10,111. They were analyzed as using polyclonal sera which comprised various antigens specific or nonspecific to T. pallidurn. If monoclonal antibodies reactive to specific antigens are used, further development would be promising in antigenic analysis of T. pallidurn. Because dark field examination is only applicable in cases of chancre which are nonreactive in the serologic test for syphilis, manipulation would be difficult or even indiscernible whether or not the organism is nonpathogenic. If a monoclonal antibody specific to T. pallidum is available, syphilitic chancre, secondary and tertiary syphilitic lesions can be diagnosed easily [ 121. Therefore, this study attempted to produce and characterize the monoclonal antibodies specific for T. pallidum. Materials and Methods Organisms T. pallidum (Nichols)
and T. phagedenis, biotype Reiter were provided by the Center for Disease Control, Atlanta, GA. T. pallidurn was passed intratesticularly in rabbits every 10 days as previously described. T, pallidum was inoculated into the testes of white rabbits with negative VDRL and TPHA in a concentration of 2-3 x 10’ T. pallidurn per testis. The infected rabbits were sacrificed on day 10, and the testes were removed. The testes were sliced, and the organisms were extracted in a solution containing 10 ml of phosphate-buffered saline (PBS) for 10 min with agitation. The solution without testicular tissue
debris was centrifuged at 1000 x g for 5 min, and the organisms in the supernatant were purified by Percoll density gradient centifugation [ 131. T. phagedenis biotype Reiter was grown in thioglycollate broth media supplemented with 10% heat inactivated rabbit serum. T. denticola and T. vincentii were provided by Dr. J.N. Miller, Dept of Microbiology, UCLA, CA, and were grown as described above. Leptospira interrogans serovar lai and Borrelia burgdorferi were provided by Dr. S.N. Cho, Dept of Microbiology, Yonsei University, Seoul, Korea. The above organisms were used as antigens in immunization, ELISA and immunoblotting. Immunization
Female BALB/c mice, obtained from Korea Advanced Institute of Science and Technology, Seoul, Korea, were immunized by subcutaneous injection of 2-4 x 10’ purified T. pallidum in complete Freund’s adjuvant. The mice were given two booster injections of 2-4 x 10’ T. pallidum by the intraperitoneal route at 4 weeks and by an intravenous route at 5 weeks. Three days after the last booster injection, the spleens were harvested. Sera were collected and pooled for use as a positive control in subsequent tests. Preparation of spleen and myeloma cells
The spleen was obtained by cervical dislocation of the immunized mouse, minced in RPM1 1640, and a cell clump was removed by passing the cell suspension through nylon mesh. Myeloma cells were either BALB/c SP 2/O or V653. Cell hybridization
Spleen and myeloma cells were mixed (at a ratio of 2.5:1) by centrifugation at 200 xg for 10 min [ 141. After tapping the conical tube gently, polyethylene glycol 1 ml 50% (w/v in serum free RPM1 1640, M- 5000) were dropped for 1 min and 1 ml RPM1 1640 for 1 min at 37 a C. 5 ml RPM1 1640 were dropped again for 5 min and incubated for 5 min.
28
After centrifugating and gently washing, the cells were suspended to 3 x lo5 cells in each well of 100 ~1 of medium containing 2.5 x lo5 normal BALB/c spleen feeder cells in 50 ,ul of HAT medium and incubated at 37 “C in a humidified atmosphere of 5% COz. One additional feeding with HAT medium (50% substitution by volume) was given on days 2 and 4. Cloning by limiting dilution [ 151 of the cell in the antibody-producing wells was performed 3 times, and cultured fluids were screened in an ELISA test with T. pallidurn, Nichols strain as previously described. For mass production of the antibody, cloned cells were grown in large quantities for i.p. injection into pristane-primed BALB/c mice (1 x 10’ cells/mouse). Isotyping The isotype of each monoclonal antibody was determined by ELISA using mouse monoclonal isotyping kit (Hyclone Lab., Logan, UT, U.S.A.). Enzyme-linked immunosorbent assay (ELISA) An ELISA was developed for detecting the antibody to antigens of T. pallidurn, T, phagedenis biotype Reiter, T. denticola, T. vincentii, L. interrogans, B. burgdorferi and rabbit testicular material [ 161. Optimal conditions were determined by antigen and antibody titration. Normal rabbit testicular antigen was obtained from homogenized suspension of serologic test for syphilis-negative rabbit. Immediately before use, antigen primed polystyrene microtiter plates were washed 3 times with 0.05% (v/v) Tween 20 (T)-PBS (pH 7.2). One hundred ~1 of supernatant of cloned cells were incubated at room temperature. After washing 3 times with T-PBS, horseradish peroxidase (HRPO)-labelled anti-mouse immunoglobulin (DAK0 Immunochemicals, Inc., Copenhagen, Denmark) diluted with 1% normal rabbit serum were incubated for 1 h. After 3 washes with TPBS, 50 ~1 of substrate-dye mixture (0.0 18% H,O, and 0.1 mg/ml o-phenylene diamine in TPBS) were added to each well. Incubation was continued in the dark at room temperature for 30 min. The reaction was stopped by the addition
of 25 ~1 of 8 N H,S04 per well. The optical density (OD,,,,,) of each well was determined on an ELISA autoreader (Dynatech Laboratories, Inc., Alexandria, VA). T. pallidurn immunized polyclonal mouse serum was used as positive control serum, normal mouse serum being used as negative. Indirect immunojluorescence Purified T. pallida, Nichols strain, were applied to glass slides, incubating with diluted ascitic fluids for 30 min. 150 diluted FITC-labelled antimouse Ig (Zymed, San Fransisco, CA) was applied to each antigen spot, and incubated for 30 min. After washing, slides were examined with a Zeiss fluorescence microscope (Carl Zeiss, Germany). Hemagglutination Produced monoclonal antibodies were detected by the ability or inability to agglutinate sheep erythrocytes sensitized with T. pallidurn, using TPHA kit (Fujirebio Pharmaceutical Co., Tokyo, Japan). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) All prepared samples were solubilized in 20 ~1 of sample buffer containing 0.5 ml sample dissolving buffer, 0.5 ml distilled water, 50 ~1 /?mercaptoethanol, 150 mg sucrose, and a trace of bromophenol blue, and were boiled for 5-10 min. After centrifugation for 5 min, the supernatants were loaded into each well for electrophoresis. The sample dissolving buffer was made of 0.15 M Tris (pH 6.8) and 4.6% sodium dodecyl sulfate. The prepared proteins were electrophoresed on 10% polyacrylamide gels in the discontinuous Tris-glycine system as described by Laemmli [ 171. Immunoblotting Electrophoretic transfer of proteins to nitrocellulose paper was performed by the method of Towbin et al. [ 181 using an electrophoretic transfer apparatus (Hoeffer Scientific Instruments, San
29 TABLE
I
Isotypes
of 7 monoclonal
TABLE antibodies
II
Reactivity sured
Clones
Isotype
YS 75, 343,406
IgG,
of anti-T. pallidurn
by ELISA,
TPHA
monoclonal
ELISA YS 3. 307,481
Ig%
YS 1
Ig%
TABLE
TPHA (titer)
(O.D.)
mea-
FTA-AB (titer)
YS 3”
0.59
640- 1280
1280
YS 75”
1.17
320-1280
41,120
YS 307”
1.33 1.40
640
640-1280
640
1.37
t80 2.0
control
Negative
a Supernatant. WR: weak reaction;
Positive
10,280
10,240
> 82,240 2.0 _
>2.0 _
0.79 -
0.32 -
Positive controls” Negative controlb
-: Nonreactive (optical density a Consists of polyvalent mouse mal rabbit testicle. b Normal mouse serum.
and
by ELISA
of less than 0.20). immunized sera to T. phagedenis,
T. denticola.
T. vincentii, L. interrogans,
rabbit
> 2.0 -
B. burgdorfri
and nor-
30
Fig. 1. SDS-PAGE
and immunoblotting
patterns
of T. pallidurn reacted
T. pallidurn; IS: polyclonal
have been grown on an inverted microscope 5 to 7 days after hybridization. Limiting dilutions were not done until the well was l/3-1/2 filled. Efficacy of fusion was rated as more than 90 %. The 7 clones (YS 3, YS 75, YS 307, YS 481, YS 343, YS 1 and YS 406) secreting monoclonal antibodies reactive with T. pallidurn were produced after cloning. The isotypes of the 7 monoclonal antibodies produced were IgG, (YS 75, 343, 406), IgG,, (YS 3, 307, 481) and IgG,, (YS 1) (Table I). Optical densities of the seven monoclonal antibodies were ranged from 0.59 to 1.48 as measured by ELISA (Table II). Monclonal antibodies from five (YS 3, 75, 307, 481, 406) of the 7 clones could agglutinate sheep erythrocyte sensitized with T. pallidurn and showed strong fluorescences, but the monoclonal antibodies from the two clones (YS 343, 1) could not agglutinate and showed very weak fluorescences (Table II). Spehjicity of monoclonal antibody The monoclonal antibody from YS 343 was cross-reactive with nonpathogenic treponemes (T. phagedenis, T. denticola, T. vincentii) but was not cross-reactive with L. interrogans, B. burgdor-
mouse
with 7 monoclonal
antiserum
antibodies.
SP: standard
protein;
TP:
to T. pallidum.
feri and normal rabbit testicular tissue by ELISA. The other 6 monoclonal antibodies reacted with T. pallidurn specifically (Table III). Molecular specificities About 45 peptides could be identified after SDS-PAGE and Coomassie brilliant blue stain. Separated antigens were reacted with each monoclonal antibody on blot and their molecular weights were determined by standard M marker protein. Monoclonal antibodies from 5 (YS 75, 307, 48 1, 1, 406) of the 7 clones reacted with a polypeptide of an M- of 47 kDa, and monoclonal antibodies from YS 343 reacted with a polypeptide of 64 kDa common to both T. pallidurn and T. phagedenis (Figs. 1,2).
Discussion
Antigenic structures of T. pallidurn have been analyzed by SDS-PAGE and immunoblotting for the understandings of pathogenic mechanisms of syphilis, and progressively by monoclonal antibodies specific for T. pallidurn. Several researchers had produced monoclonal antibodies specific
31
patterns of T. pallidurn and those of T. phagedenis reacted with 7 monoclonal Fig. 2. Cornpal :ison between immunoblotting bodies. SP: standard protein; TP: T. pallidurn; IS: polyclonal mouse antiserum to T. pallidurn.
to T. pallidurn and cross-reactive with nonpathogens or normal rabbit testicular tissue [ 19,201. We produced 7 monoclonal antibodies reacting with T. pallidum, among which the YS 343 monoclonal antibody was cross-reactive with T. phagedenis, T, denticola and T. vincentii, but the other 6 monoclonal antibodies reacted with T. pallidurn specifically. Monoclonal antibodies from two clones (Y S 343, 1) could not agglutinate sheep erythrocyte sensitized with T. pallidurn and showed very weak fluorescence. This result might have been due to a difference in antibody measuring methods or cross-reactivity with T. phagedenis.
Treponemal antigen of 47 kDa is an immunodominant surface associated polypeptide [ 2 1] which showed strong immunogenicity in T. pallidum infected humans and rabbits. Our results in which 5 of 7 monoclonal antibodies reacted with a polypeptide of 47 kDa, also suggest an immunodominancy of 47 kDa antigen. Bailey et al. [2] demonstrated that antigens of 44 and 47 kDa were outer membrane associated polypeptides of T. pallidum and that only antibodies directed against the 44 kDa polypeptide had immobilizing activity. Polypeptide of 47 kDa is known to have
anti-
a common antigen with T. phagedenis [6]; but, Lukehart et al. [ 31 contended that polypeptide of 48 kDa was specific to T. pallidurn, and that decreased reactivity after absorbing syphilitic serum with T. phagedenis was because T. pallidum specific and common antigenic epitopes are included in the same molecular polypetide. This polypeptide of 48 kDa might be considered the same as the polypeptide of 47 kDa in our study. The above suggestion is supported by the fact that monoclonal antibodies reacting to 47 kDa molecule did not show any other bands on immunoblotting. So, these antibodies reacting to 47 kDa might be specific to T. pallidum. The fact that this molecule can also be found in normal rabbit testicular tissue and normal rabbit serum [ 111 suggests that these materials contaminate to treponemal antigens preparations or treponemal antigens have similar electrophoretic mobility to the tissue protein antigens of a rabbit [ 31. A comparison of the reactivity between monoclonal antibodies of M.V. Nogard [ 221 and those of Bailey et al. [2] on T. pallidurn antigenic profiles revealed different patterns on immunoblot. These findings suggest that a variety of epitopes may be present on this surface associated poly-
32
peptide. Even though some monoclonal antibodies showed same or different isotypic monoclonal antibodies, we could not define whether these reacted with same epitope or not. YS 3 monoclonal antibody did not show any bands even though immunoblottings had been tried several times. We do not know why exactly. But we guess it is because SDS-PAGE breaks down the antigenic proteins, and that antibody shows reactivities only in case of using serologic methods. Nonpathogenic treponemes can be cultured in thioglycollate broth media, but T. pallidurn can only be cultured by inoculation into a rabbit testicle, so, to obtain relatively large amounts requires much time and expense. Recombinant DNA clones expressing T. pallidurn gene encoding for 47 kDa antigen might be useful as an antigen in serologic tests for syphilis. We plan to screen the recombinant DNA clones and to use the result as a diagnostic application along with ELISA-inhibition test and immunoscintigraphy by using our monoclonal antibodies specific to T. pallidum.
9
10
11
12
13
14
15
16
References 1 Sell S, Norris SJ: The biology, pathology and immunology of syphilis. Int Rev Exp Path01 24: 203, 1983. 2 Bailey MJ, Cockayne A, Penn W: Monoclonal antibodies directed against surface-associated polypeptides of Treponema pallidurn define a biologically active antigen. J Microbial 133: 1793, 1987. 3 Lukehart SA, Baker-Zander SA, Gubish ER Jr.: Identification of Treponema pallidum antigens: Comparison with a nonpathogenic treponeme. J Immunol 129: 833, 1982. 4 Hanff PA, Fehniger TE, Miller JN, et al.: Humoral immune response in human syphilis to polypeptides of Treponemapallidum. J Immunol 129: 1287, 1982. 5 Hanff PA, Bishop NH, Miller JN, et al.: Humoral immune response in experimental syphilis to polypeptides of Treponema pallidurn. J Immunol 13 1: 1973, 1983. 6 Hanff PA, Miller JN, Lovett MA: Molecular characterization of common treponemal antigens. Infect Immun 40: 825, 1983. 7 Moskophidis M, Muller F: Molecular analysis of immunoglobulins M and G immune response to protein antigens of Treponema palfidum in human syphilitic sera. Infect Immun 43: 127, 1984. 8 Petersen CS, Pedersen NS, Axelsen NH: Purification of
17
18
19
20
21
22
23
TR-b, a Reiter treponeme protein antigen precipitating with antibodies in human syphilitic sera. Infect Immun 38: 35, 1982. Wither K, Wos SM, Wither V: Kinetics of antibody response to polypeptides of pathogenic and nonpathogenic treponemes in experimental syphilis. Sex Transm Dis 13: 251, 1986. Lee MG, Sung YO, Kim DK, et al.: Protein antigens of Treponema pallidum reacting with serum Ig M antibodies of syphilis patients. Kor J Dermatol 27: 362, 1989. Kim DK, Lee MG, Lee JB: Changes of serum IgG antibody reactivity to protein antigens of Treponema pallidurn in syphilis patients after treatment. J Kor Med Sci 4: 63, 1989. Kohler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495, 1975. Hanff PA, Norris SJ, Lovett MA, et al.: Purification of Treponemapallidum, Nichols strain, by percoll density gradient centrifugation. Sex Transm Dis 11: 275, 1984. Galfre G, Howe SC, Milstein C, et al.: Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266: 550, 1977. McKearn TJ: Cloning of hybridoma cells by limiting dilution in fluid phase 374, in RH Kennet, TJ Mckearn, KB Bechtol. Edited by Monoclonal Antibodies, Hybridoma: A Mew Dimension in Biological Analysis. Edited by Plenum Press, New York, 1980. Lee MG, Han SW, Lee JB: Detection of immunoglobulin M antibodies to Treponema pallidum in sera from syphilis patients by enzyme-linked immunosorbent assay. Yonsei J Med Sci 20: 379, 1987. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680, 1970. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat1 Acad Sci USA 76: 4350, 1979. Robertson SM, Kettman JR. Miller JN, et al.: Murine monoclonal antibodies specific for virulent Treponema pallidurn (Nichols). Infect Immun 36: 1076, 1982. Lukehart SA, Tam MR, Horn. et al.: Characterization of monoclonal antibodies to Treponema pallidurn. J Immunol 134: 585, 1985. Penn CW, Cockayne A, Bailey MJ: The outer membrane of Treponema pallidurn: Biological significance and biological properties. J Gen Microbial 131: 2249, 1985. Marchitto KS, Jones SA, Schell RF, et al.: Monoclonal antibody analysis of specific antigenic similarities among pathogenic Treponemapallidum species. Infect Immun 45: 660, 1984. Lowry OH, Rosebrough NJ, Farr AL, Randall R: Protein measurement with the folin phenol reagent. J Biol Chem 193: 265, 1951.
26
DESC
1992 Elsevier
Science
Publishers
B.V. All rights
reserved.
0923-181
l/92/$05.00
00148
Production
and characterization
of monoclonal
antibodies to Treponema
pallidurn Kyu Kwang
Whang,
Min Geol
Lee ‘, Wook
Lew ’ and Jung Bock
Departments of Dermatology. Konkuk University College of Medicine, Chungkuk, Korea and
Lee ’
’ Yonsei Universitv College of Medicine,
Seoul. Korea (Received
13 December
Key words:
1990; accepted
Monoclonal
27 December
1991)
Treponema pallidurn
antibody;
Abstract This study
was attempted
to produce
the monoclonal
antibodies
for Treponema pallidum and to investigate
specific
teristics, thereby contributing to identify the antigenic structure and to apply the diagnosis YS 75, YS 307, YS 481, YS 343, YS 1 and YS 406) secreting monoclonal antibodies reactive isotypes of the seven monoclonal from 0.59 to 1.48 as measured sensitized pathogenic clonal
with
T. pallidurn, showed
treponemes,
antibodies
antibodies by ELISA.
from
ies from YS 343 reacted
but the other
strong
produced were defined. Monclonal antibodies fluorescences.
6 monoclonal
5 of the 7 clones with a polypeptide
reacted
Optical densities of the 7 monoclonal from 5 of the 7 clones which could
The monoclonal
antibodies
reacted
with a polypeptide
of 64 kDa common
antibody
to both
antibodies agglutinate
were ranged sheep RBC
from YS 343 was cross-reactive
with T. pa/lidum specifically.
of a molecular
their charac-
of syphilis. The seven clones (YS 3, with T. pallidzrm were produced. The
weight
of 47 kDa,
On immunoblotting. and monoclonal
T. pallidum and T. phagedenis. The above results
with nonmonoantibodrevealed
that 7 clones secreting monoclonal antibodies reactive to T. pallidum were produced successfully, and specific antibodies reacting with major antigenic polypeptides of a molecular weight of 47 kDa would be useful in the diagnosis of syphilis in the future.
Introduction Treponema pallidurn, T. pertenue and T. carateum inducing syphilis, yaw and pinta, respectively, are included among pathogenic treponemes; T. denticola, T. microdentium, and T. phagedenis, T. refringens, and T. vincentii, which reside normally in oral, genital, and gastroenteric mucosa, are included among nonpathoCorrespondence to: J.B. Lee, Department of Dermatology, Yonsei University College of Medicine, CPO Box 8044, Seoul, Korea.
genie treponemes [ 11. Nonpathogens can be cultured in broth thioglycollate media, but T. pallidum can be cultured only in rabbit testicle; consequently, so relatively small amounts can be obtained. For this reason, there are many restrictions in pathogenic, immunodiagnostic, and preventive studies. Therefore there have been studies of monoclonal antibodies specific for T. pallidurn and its recombinant antigenic cloning genetically [2]. But these should be preceded by biological and biochemical identifications of polypeptides of T. pallidurn. Those polypeptides were separated on sodium
21
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were electrophoretically transferred to a nitrocellulose paper for antigenic analysis. Eight to 23 protein antigens of T. pallidum are identifiable today [ 3-71. The number of common antigens with T. phagedenis were reported as 5,6, 8, 11 or 15, differing according to authors and measuring methods [ 3,6,8,9]. We have observed that immunodominant antigens were polypeptides of 47, 36.5, 29.5, 15.5 and 14 kDa, and that 11 antigens were common to both T. palhdum and T. phagedenis [ 10,111. They were analyzed as using polyclonal sera which comprised various antigens specific or nonspecific to T. pallidurn. If monoclonal antibodies reactive to specific antigens are used, further development would be promising in antigenic analysis of T. pallidurn. Because dark field examination is only applicable in cases of chancre which are nonreactive in the serologic test for syphilis, manipulation would be difficult or even indiscernible whether or not the organism is nonpathogenic. If a monoclonal antibody specific to T. pallidum is available, syphilitic chancre, secondary and tertiary syphilitic lesions can be diagnosed easily [ 121. Therefore, this study attempted to produce and characterize the monoclonal antibodies specific for T. pallidum. Materials and Methods Organisms T. pallidum (Nichols)
and T. phagedenis, biotype Reiter were provided by the Center for Disease Control, Atlanta, GA. T. pallidurn was passed intratesticularly in rabbits every 10 days as previously described. T, pallidum was inoculated into the testes of white rabbits with negative VDRL and TPHA in a concentration of 2-3 x 10’ T. pallidurn per testis. The infected rabbits were sacrificed on day 10, and the testes were removed. The testes were sliced, and the organisms were extracted in a solution containing 10 ml of phosphate-buffered saline (PBS) for 10 min with agitation. The solution without testicular tissue
debris was centrifuged at 1000 x g for 5 min, and the organisms in the supernatant were purified by Percoll density gradient centifugation [ 131. T. phagedenis biotype Reiter was grown in thioglycollate broth media supplemented with 10% heat inactivated rabbit serum. T. denticola and T. vincentii were provided by Dr. J.N. Miller, Dept of Microbiology, UCLA, CA, and were grown as described above. Leptospira interrogans serovar lai and Borrelia burgdorferi were provided by Dr. S.N. Cho, Dept of Microbiology, Yonsei University, Seoul, Korea. The above organisms were used as antigens in immunization, ELISA and immunoblotting. Immunization
Female BALB/c mice, obtained from Korea Advanced Institute of Science and Technology, Seoul, Korea, were immunized by subcutaneous injection of 2-4 x 10’ purified T. pallidum in complete Freund’s adjuvant. The mice were given two booster injections of 2-4 x 10’ T. pallidum by the intraperitoneal route at 4 weeks and by an intravenous route at 5 weeks. Three days after the last booster injection, the spleens were harvested. Sera were collected and pooled for use as a positive control in subsequent tests. Preparation of spleen and myeloma cells
The spleen was obtained by cervical dislocation of the immunized mouse, minced in RPM1 1640, and a cell clump was removed by passing the cell suspension through nylon mesh. Myeloma cells were either BALB/c SP 2/O or V653. Cell hybridization
Spleen and myeloma cells were mixed (at a ratio of 2.5:1) by centrifugation at 200 xg for 10 min [ 141. After tapping the conical tube gently, polyethylene glycol 1 ml 50% (w/v in serum free RPM1 1640, M- 5000) were dropped for 1 min and 1 ml RPM1 1640 for 1 min at 37 a C. 5 ml RPM1 1640 were dropped again for 5 min and incubated for 5 min.
28
After centrifugating and gently washing, the cells were suspended to 3 x lo5 cells in each well of 100 ~1 of medium containing 2.5 x lo5 normal BALB/c spleen feeder cells in 50 ,ul of HAT medium and incubated at 37 “C in a humidified atmosphere of 5% COz. One additional feeding with HAT medium (50% substitution by volume) was given on days 2 and 4. Cloning by limiting dilution [ 151 of the cell in the antibody-producing wells was performed 3 times, and cultured fluids were screened in an ELISA test with T. pallidurn, Nichols strain as previously described. For mass production of the antibody, cloned cells were grown in large quantities for i.p. injection into pristane-primed BALB/c mice (1 x 10’ cells/mouse). Isotyping The isotype of each monoclonal antibody was determined by ELISA using mouse monoclonal isotyping kit (Hyclone Lab., Logan, UT, U.S.A.). Enzyme-linked immunosorbent assay (ELISA) An ELISA was developed for detecting the antibody to antigens of T. pallidurn, T, phagedenis biotype Reiter, T. denticola, T. vincentii, L. interrogans, B. burgdorferi and rabbit testicular material [ 161. Optimal conditions were determined by antigen and antibody titration. Normal rabbit testicular antigen was obtained from homogenized suspension of serologic test for syphilis-negative rabbit. Immediately before use, antigen primed polystyrene microtiter plates were washed 3 times with 0.05% (v/v) Tween 20 (T)-PBS (pH 7.2). One hundred ~1 of supernatant of cloned cells were incubated at room temperature. After washing 3 times with T-PBS, horseradish peroxidase (HRPO)-labelled anti-mouse immunoglobulin (DAK0 Immunochemicals, Inc., Copenhagen, Denmark) diluted with 1% normal rabbit serum were incubated for 1 h. After 3 washes with TPBS, 50 ~1 of substrate-dye mixture (0.0 18% H,O, and 0.1 mg/ml o-phenylene diamine in TPBS) were added to each well. Incubation was continued in the dark at room temperature for 30 min. The reaction was stopped by the addition
of 25 ~1 of 8 N H,S04 per well. The optical density (OD,,,,,) of each well was determined on an ELISA autoreader (Dynatech Laboratories, Inc., Alexandria, VA). T. pallidurn immunized polyclonal mouse serum was used as positive control serum, normal mouse serum being used as negative. Indirect immunojluorescence Purified T. pallida, Nichols strain, were applied to glass slides, incubating with diluted ascitic fluids for 30 min. 150 diluted FITC-labelled antimouse Ig (Zymed, San Fransisco, CA) was applied to each antigen spot, and incubated for 30 min. After washing, slides were examined with a Zeiss fluorescence microscope (Carl Zeiss, Germany). Hemagglutination Produced monoclonal antibodies were detected by the ability or inability to agglutinate sheep erythrocytes sensitized with T. pallidurn, using TPHA kit (Fujirebio Pharmaceutical Co., Tokyo, Japan). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) All prepared samples were solubilized in 20 ~1 of sample buffer containing 0.5 ml sample dissolving buffer, 0.5 ml distilled water, 50 ~1 /?mercaptoethanol, 150 mg sucrose, and a trace of bromophenol blue, and were boiled for 5-10 min. After centrifugation for 5 min, the supernatants were loaded into each well for electrophoresis. The sample dissolving buffer was made of 0.15 M Tris (pH 6.8) and 4.6% sodium dodecyl sulfate. The prepared proteins were electrophoresed on 10% polyacrylamide gels in the discontinuous Tris-glycine system as described by Laemmli [ 171. Immunoblotting Electrophoretic transfer of proteins to nitrocellulose paper was performed by the method of Towbin et al. [ 181 using an electrophoretic transfer apparatus (Hoeffer Scientific Instruments, San
29 TABLE
I
Isotypes
of 7 monoclonal
TABLE antibodies
II
Reactivity sured
Clones
Isotype
YS 75, 343,406
IgG,
of anti-T. pallidurn
by ELISA,
TPHA
monoclonal
ELISA YS 3. 307,481
Ig%
YS 1
Ig%
TABLE
TPHA (titer)
(O.D.)
mea-
FTA-AB (titer)
YS 3”
0.59
640- 1280
1280
YS 75”
1.17
320-1280
41,120
YS 307”
1.33 1.40
640
640-1280
640
1.37
t80 2.0
control
Negative
a Supernatant. WR: weak reaction;
Positive
10,280
10,240
> 82,240 2.0 _
>2.0 _
0.79 -
0.32 -
Positive controls” Negative controlb
-: Nonreactive (optical density a Consists of polyvalent mouse mal rabbit testicle. b Normal mouse serum.
and
by ELISA
of less than 0.20). immunized sera to T. phagedenis,
T. denticola.
T. vincentii, L. interrogans,
rabbit
> 2.0 -
B. burgdorfri
and nor-
30
Fig. 1. SDS-PAGE
and immunoblotting
patterns
of T. pallidurn reacted
T. pallidurn; IS: polyclonal
have been grown on an inverted microscope 5 to 7 days after hybridization. Limiting dilutions were not done until the well was l/3-1/2 filled. Efficacy of fusion was rated as more than 90 %. The 7 clones (YS 3, YS 75, YS 307, YS 481, YS 343, YS 1 and YS 406) secreting monoclonal antibodies reactive with T. pallidurn were produced after cloning. The isotypes of the 7 monoclonal antibodies produced were IgG, (YS 75, 343, 406), IgG,, (YS 3, 307, 481) and IgG,, (YS 1) (Table I). Optical densities of the seven monoclonal antibodies were ranged from 0.59 to 1.48 as measured by ELISA (Table II). Monclonal antibodies from five (YS 3, 75, 307, 481, 406) of the 7 clones could agglutinate sheep erythrocyte sensitized with T. pallidurn and showed strong fluorescences, but the monoclonal antibodies from the two clones (YS 343, 1) could not agglutinate and showed very weak fluorescences (Table II). Spehjicity of monoclonal antibody The monoclonal antibody from YS 343 was cross-reactive with nonpathogenic treponemes (T. phagedenis, T. denticola, T. vincentii) but was not cross-reactive with L. interrogans, B. burgdor-
mouse
with 7 monoclonal
antiserum
antibodies.
SP: standard
protein;
TP:
to T. pallidum.
feri and normal rabbit testicular tissue by ELISA. The other 6 monoclonal antibodies reacted with T. pallidurn specifically (Table III). Molecular specificities About 45 peptides could be identified after SDS-PAGE and Coomassie brilliant blue stain. Separated antigens were reacted with each monoclonal antibody on blot and their molecular weights were determined by standard M marker protein. Monoclonal antibodies from 5 (YS 75, 307, 48 1, 1, 406) of the 7 clones reacted with a polypeptide of an M- of 47 kDa, and monoclonal antibodies from YS 343 reacted with a polypeptide of 64 kDa common to both T. pallidurn and T. phagedenis (Figs. 1,2).
Discussion
Antigenic structures of T. pallidurn have been analyzed by SDS-PAGE and immunoblotting for the understandings of pathogenic mechanisms of syphilis, and progressively by monoclonal antibodies specific for T. pallidurn. Several researchers had produced monoclonal antibodies specific
31
patterns of T. pallidurn and those of T. phagedenis reacted with 7 monoclonal Fig. 2. Cornpal :ison between immunoblotting bodies. SP: standard protein; TP: T. pallidurn; IS: polyclonal mouse antiserum to T. pallidurn.
to T. pallidurn and cross-reactive with nonpathogens or normal rabbit testicular tissue [ 19,201. We produced 7 monoclonal antibodies reacting with T. pallidum, among which the YS 343 monoclonal antibody was cross-reactive with T. phagedenis, T, denticola and T. vincentii, but the other 6 monoclonal antibodies reacted with T. pallidurn specifically. Monoclonal antibodies from two clones (Y S 343, 1) could not agglutinate sheep erythrocyte sensitized with T. pallidurn and showed very weak fluorescence. This result might have been due to a difference in antibody measuring methods or cross-reactivity with T. phagedenis.
Treponemal antigen of 47 kDa is an immunodominant surface associated polypeptide [ 2 1] which showed strong immunogenicity in T. pallidum infected humans and rabbits. Our results in which 5 of 7 monoclonal antibodies reacted with a polypeptide of 47 kDa, also suggest an immunodominancy of 47 kDa antigen. Bailey et al. [2] demonstrated that antigens of 44 and 47 kDa were outer membrane associated polypeptides of T. pallidum and that only antibodies directed against the 44 kDa polypeptide had immobilizing activity. Polypeptide of 47 kDa is known to have
anti-
a common antigen with T. phagedenis [6]; but, Lukehart et al. [ 31 contended that polypeptide of 48 kDa was specific to T. pallidurn, and that decreased reactivity after absorbing syphilitic serum with T. phagedenis was because T. pallidum specific and common antigenic epitopes are included in the same molecular polypetide. This polypeptide of 48 kDa might be considered the same as the polypeptide of 47 kDa in our study. The above suggestion is supported by the fact that monoclonal antibodies reacting to 47 kDa molecule did not show any other bands on immunoblotting. So, these antibodies reacting to 47 kDa might be specific to T. pallidum. The fact that this molecule can also be found in normal rabbit testicular tissue and normal rabbit serum [ 111 suggests that these materials contaminate to treponemal antigens preparations or treponemal antigens have similar electrophoretic mobility to the tissue protein antigens of a rabbit [ 31. A comparison of the reactivity between monoclonal antibodies of M.V. Nogard [ 221 and those of Bailey et al. [2] on T. pallidurn antigenic profiles revealed different patterns on immunoblot. These findings suggest that a variety of epitopes may be present on this surface associated poly-
32
peptide. Even though some monoclonal antibodies showed same or different isotypic monoclonal antibodies, we could not define whether these reacted with same epitope or not. YS 3 monoclonal antibody did not show any bands even though immunoblottings had been tried several times. We do not know why exactly. But we guess it is because SDS-PAGE breaks down the antigenic proteins, and that antibody shows reactivities only in case of using serologic methods. Nonpathogenic treponemes can be cultured in thioglycollate broth media, but T. pallidurn can only be cultured by inoculation into a rabbit testicle, so, to obtain relatively large amounts requires much time and expense. Recombinant DNA clones expressing T. pallidurn gene encoding for 47 kDa antigen might be useful as an antigen in serologic tests for syphilis. We plan to screen the recombinant DNA clones and to use the result as a diagnostic application along with ELISA-inhibition test and immunoscintigraphy by using our monoclonal antibodies specific to T. pallidum.
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10
11
12
13
14
15
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References 1 Sell S, Norris SJ: The biology, pathology and immunology of syphilis. Int Rev Exp Path01 24: 203, 1983. 2 Bailey MJ, Cockayne A, Penn W: Monoclonal antibodies directed against surface-associated polypeptides of Treponema pallidurn define a biologically active antigen. J Microbial 133: 1793, 1987. 3 Lukehart SA, Baker-Zander SA, Gubish ER Jr.: Identification of Treponema pallidum antigens: Comparison with a nonpathogenic treponeme. J Immunol 129: 833, 1982. 4 Hanff PA, Fehniger TE, Miller JN, et al.: Humoral immune response in human syphilis to polypeptides of Treponemapallidum. J Immunol 129: 1287, 1982. 5 Hanff PA, Bishop NH, Miller JN, et al.: Humoral immune response in experimental syphilis to polypeptides of Treponema pallidurn. J Immunol 13 1: 1973, 1983. 6 Hanff PA, Miller JN, Lovett MA: Molecular characterization of common treponemal antigens. Infect Immun 40: 825, 1983. 7 Moskophidis M, Muller F: Molecular analysis of immunoglobulins M and G immune response to protein antigens of Treponema palfidum in human syphilitic sera. Infect Immun 43: 127, 1984. 8 Petersen CS, Pedersen NS, Axelsen NH: Purification of
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TR-b, a Reiter treponeme protein antigen precipitating with antibodies in human syphilitic sera. Infect Immun 38: 35, 1982. Wither K, Wos SM, Wither V: Kinetics of antibody response to polypeptides of pathogenic and nonpathogenic treponemes in experimental syphilis. Sex Transm Dis 13: 251, 1986. Lee MG, Sung YO, Kim DK, et al.: Protein antigens of Treponema pallidum reacting with serum Ig M antibodies of syphilis patients. Kor J Dermatol 27: 362, 1989. Kim DK, Lee MG, Lee JB: Changes of serum IgG antibody reactivity to protein antigens of Treponema pallidurn in syphilis patients after treatment. J Kor Med Sci 4: 63, 1989. Kohler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495, 1975. Hanff PA, Norris SJ, Lovett MA, et al.: Purification of Treponemapallidum, Nichols strain, by percoll density gradient centrifugation. Sex Transm Dis 11: 275, 1984. Galfre G, Howe SC, Milstein C, et al.: Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266: 550, 1977. McKearn TJ: Cloning of hybridoma cells by limiting dilution in fluid phase 374, in RH Kennet, TJ Mckearn, KB Bechtol. Edited by Monoclonal Antibodies, Hybridoma: A Mew Dimension in Biological Analysis. Edited by Plenum Press, New York, 1980. Lee MG, Han SW, Lee JB: Detection of immunoglobulin M antibodies to Treponema pallidum in sera from syphilis patients by enzyme-linked immunosorbent assay. Yonsei J Med Sci 20: 379, 1987. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680, 1970. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat1 Acad Sci USA 76: 4350, 1979. Robertson SM, Kettman JR. Miller JN, et al.: Murine monoclonal antibodies specific for virulent Treponema pallidurn (Nichols). Infect Immun 36: 1076, 1982. Lukehart SA, Tam MR, Horn. et al.: Characterization of monoclonal antibodies to Treponema pallidurn. J Immunol 134: 585, 1985. Penn CW, Cockayne A, Bailey MJ: The outer membrane of Treponema pallidurn: Biological significance and biological properties. J Gen Microbial 131: 2249, 1985. Marchitto KS, Jones SA, Schell RF, et al.: Monoclonal antibody analysis of specific antigenic similarities among pathogenic Treponemapallidum species. Infect Immun 45: 660, 1984. Lowry OH, Rosebrough NJ, Farr AL, Randall R: Protein measurement with the folin phenol reagent. J Biol Chem 193: 265, 1951.
