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Production of Monoclonal Antibodies Against Leptospiral Genus-Specific Protein Antigen and Localization of the Antigen by Immunoelectron Microscopy
Zbl. Bakt. Hyg. A 259, 557-563 (1985)
Production of Monoclonal Antibodies Against Leptospiral Genus-Specific Protein Antigen and Localization of the Antigen by Immunoe1ectron Microscopy NORITSUGU SAKAMOTO, ETSURO ONO, HIROSHI KIDA, and RYO YANAGAWA Department of Hygiene and Microbiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan (Head: Prof. Dr. Yanagawa) With 2 Figures' Received January 14, 1985 . Accepted February 2, 1985
Abstract Monoclonal antibodies were first produced against a leptospiral genus-specific protein antigen (GP-Ag) isolated from Leptospira interrogans serovar kremastos strain Kyoto. The representative monoclonal antibody (GP-?) did not agglutinate kremastos Kyoto but reacted with Triton X-100 treated kreamastos Kyoto, andamana, patoc and Urawa in enzyme-linked immunosorbent assay (ELISA), indicating that the monoclonal antibody recognized the genus-specific determinant of GP-Ag. The localization of GP-Ag was investigated by immunoelectron microscopy with the iodinated antibody and peroxidase-labeled antibody. The genus-specific determinant of GPAg recognized by GP-? was located on the subsurface of the leptospiral envelope. Zusammenfassung Monoklonale Antikorper wurden zunachsr gegen ein genus-spezifisches Protein-Antigen (GP-Ag) gebildet, das aus Leptospira interrogans, serovar kremastos, Stamm Kyoto isoliert worden war. Der reprasentative monoklonale Antikorper (GP-?) agglutinierte kremastos Kyoto nicht, reagierte aber mit Triton-X-lOO-behandeiten kremastos Kyoto, andamana, patoc und Urawa im ELISA, was anzeigt, daf der monoklonale Antikorper die genusspezifische Determinante von GP-Ag erkannte. Die Lokalisation von GP-Ag wurde immunelektronenmikroskopisch mit jodierten Antikorpern und peroxidasemarkierten Antikorpern untersucht. Die durch GP-? erkannte genus-spezifische Determinante von GP-Ag war unterhalb der Oberflache der BakterienhiilIe lokalisiert.
Introduction Monoclonal antibodies against leptospiral antigens have been produced by Ono et a1. (9) and Kobayashi et a1. (7) against serovar-specific antigens, and by Adler and Paine (2) against a species-specific antigen. But up to now, no monoclonal antibodies have been produced against leptospiral genus-specific antigen.
558
N. Sakamoto, E. Ono, H. Kida, and R. Yanagawa
A partially purified leptospiral genus-specific protein antigen (GP-Ag) was isolated from Leptospira interrogans serovar kremastos strain Kyoto by treating the organisms with Triton X-lOa followed by using a purification procedure including fractionation with DEAE-cellulose column chromatography and ethanol precipitation. The molecular weight of GP-Ag was estimated to be approximately 62 000. The protein part of GP-Ag which was responsible for its genus-specificity did not appear to be located on the leptospiral cell surface, because the antiserum to GP-Ag did not agglutinate L. biflexa and the serovars of L. interrogans except those belonging to Hebdomadis serogroup (In press). Little information is available on the localization of leptospiral antigens by immunoelectron microscopy. Only one report was published by Osechinsky et al. (10), who showed that iodinated mouse anti-canicola antibodies bound to the surface of the homologous leptospiras. In the present study, a monoclonal antibody against GP-Ag was produced and the localization of GP-Ag was investigated by immunoelectron microscopy with the monoclonal antibody. Materials and Methods Production of monoclonal antibody to the leptospiral genus-specific protein antigen
GP-Ag was purified from Leptospira interrogans serovar kremastos strain Kyoto (12) grown in Shenberg's synthetic medium (11) according to Sakamoto et al. (In press). Monoclonal antibody to GP-Ag was prepared in mouse hybridorna system (6). The spleen cell donor was a BALB/c mouse subcutaneousl y and intramuscularly immunized with 0.25 ml of emulsion consisting of one part of phosphate-buffered saline (PBS, pH 7.2) containing 500 I!gGP-Ag, and one part of Freund's complete adjuvant three times at 7-day intervals. Fusion was carried out 2 months later, 3 days after an intraperitoneal booster injection of 500 ug of GP-Ag. Monoclonal antibody to the serovar-specific antigen
Monoclonal antibody (KT-1) to the serovar-specific antigen (TM antigen) of kremastos strain Kyoto produced by Ono et al. (9) was used. Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed virtually according to the method described (6). Triton X-lOOtreated kremastos Kyoto, andamana, patoc and Urawa were used as coating antigen. Microscopic agglutination test
Microscopic agglutination test was done as reported (4). Iodinated antibody and horseradish peroxidase-labeled antibody
IgG-fractions from mouse ascitic fluids purified by the method of Ey et al. (5) were iodinated by the method of Mekler et al. (8) and labeled with horseradish peroxidase (Sigma Chern. Co., St. Louis, USA; EC 1.11.1.7) by the method of Avrameas (3). Immunoelectron microscopic technique
Immunoelectron microscopic analysis using iodinated monoclonal antibody was done according to Mekler et al. (8). Samples were examined under a Hitachi HU-12A electron microscope.
Localization of Leptospiral Genus-Specific Antigen
559
Immunoelectron microscopic analysis using horseradish peroxidase (HRPO)-labeled antibody was done as follows. Leptospiral cells in 10 ml of Shenberg's synthetic medium were incubated for 3 h at room temperature with 100 III of the HRPO-Iabeled antibody preparat ions. A hundred III of substrate solution (0.05 M Tris-HCl buffer, pH 7.6, 6 mM 3.3'-diaminobenzidine-tetrahydrochloride, 0.05 %, vlv, hydrogen peroxide) were added, and this solution was incubated for 30 min at room temperature. The material was fixed in OS04, dehydrated and embedded in Epon. Ultrathin sections were examined under a Hitachi HU-12A electron microscope. Results
Reactivities of mon oclonal antibodies to GP-Ag Four hybridoma cell lines were established against GP-Ag. Of these, GP-7 did not agglut inate the homologous kremastos strain Kyoto in microscopic agglutination test (negative at 1: 10) (Ta ble 1). GP-7 reacted with kremastos Kyoto of L. interrogans, and andaman a, patoc and Urawa of L. biflexa in ELISA. The remaining 3 antibodies gave similar results. KT-1, which was a monoclonal antibody to TM antigen , agglutinated kremastos in microscopi c agglutination test, and reacted with kremastos but not with patoc in ELISA.
lmmunoelectron microscopy with iodinated monoclonal antibody No iodin ated GP-7 was bound to the lepto spiral cell (Fig. 1a). On the other hand, iodinated KT-1 was bound to the surface of the lepto spiral cell (Fig. 1b). Thu s, it was consid ered that the antigenic determin ant of GP-Ag recognized by GP-7 was not located on the surface of the lepto spiral envelope, while the ant igenic determin ant of the TM anti gen recognized by KT-I was located on the leptospiral cell surface. Figure l c shows control organisms with no added iodinated antibody.
lmmunoeleetron microscopy with HR PO-Iabeled monoclonal antib ody No labeled GP-7 was bound to the lepto spiral cell (Fig. 2a). Labeled GP-7 was bound to the lepto spiral envelope only when the lepto spiral cells were dam aged and the envelope was released from its protoplasmic cylinder (Fig. 2b). On the other hand, labeled KT-1 was bound to the surface of the leptospiral cell (Fig. 2c). Figure 2d shows control organisms with no add ed HRPO-I abeled antib ody. Thus, it was considered that the ant igenic determin ant of GP-Ag recognized by GP-7 was located on the sub surface of the lepto spiral envelope, while th e antigenic determ inant of the TM antigen recognized by KT-1 was located on the leptospiral cell surface. Discussion Mon oclon al antibodies to the leptospiral genus-specific protein antigen (GP-Ag) were produced for the first time. The monocl onal antib odies, including GP-7, did not agglutinate kremastos Kyoto but reacted with Triton X-lO O-treated kremastos Kyoto , andam ana, patoc and Urawa in ELISA. The localization of GP-Ag by immunoelectron microscopy with GP-7 show ed that the antigenic determ inant of GP-Ag recognized by GP-7 was located on the subsurface of the leptospiral envelope . This finding does not contradict the fact th at the antiserum
102,400 409,600
< 10
128,000
ELISA wirlr' kremastos
MAT with 2 kremastos 12,800 NT 4
ELISA with andamana 102,400
< 100
ELISA with patoc
25,600 NT
ELISA with Urawa
< 100 NT
ELISA with Leptonema illini
Immunoglobulin was tested for the class of immunoglobulin light and heavy chains by double immunodiffusion using specific antisera to various kinds of myeloma protein or each light chain subclass (Miles Lab., Inc., Elkhart, Ind., USA) . Reciprocal of the dilution of monoclonal antibo dies expressed in 50% microscopic agglutination. MAT: microscopic agglutination test. Titers are expressed as the highest dilution of antibody to shows significant binding above background level. NT: not tested .
yl. x. y3. x.
GP-7 KT-1
1
Irnmunoglobulin 1 type
Monoclonal antibody
Table 1. Reactivities of monoclonal antibodies to GP-Ag (GP-7) and TM antigen (KT-1)
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~
OQ
I>l
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~
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;:r::
.0
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o
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;.;-
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;z:
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Localization of Leptospiral Genus-Specific Antigen
561
a
b
c
Fig. 1. Electronmicrographs of L. interrogans serovar kremastos strain Kyoto added with iodinated monoclonal antibody GP-7 (a), iodinated monoclonal antibody KT-1 (b), and with no added iodinated antibody (c) (x 25,000 ). 34 Zbl. Bakt. Hyg. A 25914
562
N. Sakamoto , E.Ono, H. Kida, and R. Yanagawa
.c
"
o Fig. 2. Electronmicrograph s of ultrathinsection of L. interrogans serovar kremastos strain Kyoto added with HRPO -labeled monoclonal antibody GP-? (a and b), HRPO-Iabeled monoclonal antibody KT-l (e), and with no added HRPO-Iabeled antibody (d) (x 60,000 ).
Localization of Leptospiral Genus-Specific Antigen
563
to GP-Ag did not agglutinate 1. biflexa and the serovars with the exceptions of those belonging to Hebdomadis serogroup of 1. interrogans (In press). Results of immunoelectron microscopic analysis of localization of serovar-specific (TM) antigen showed that TM antigen was located on the leptospiral cell surface. This finding agrees with the fact that TM antigen was associated with microscopic agglutination and that TM antigen inhibited leptospiral agglutination (1). The location on the subsurface of GP-Ag in the leptospiral envelope may lead to further studies on the role of GP-Ag in the architecture of the leptospiral envelope and on function of GP-Ag such as transportion of low molecular substances, etc. The use of monoclonal antibody against GP-Ag may be helpful not only in elucidating localization of GP-Ag, but also in isolating and characterizing the antigenic determinant of GP-Ag, which will be the subject of a future study.
References 1. Adachi, Y. and R. Yanagawa: Inhibition of leptospiral agglutination by the type-specific main antigens of leptospiras. Infect. Immun. 17 (1977) 466-467 2. Adler, B. and S. Paine: Species- and genus-specific antigens in leptospira, revealed by monoclonal antibodies and enzyme immunoassay. Zbl. Bakt. Hyg., I. Abt. Orig. A 255 (1983) 317-322 3. Avrameas, S.: Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry 6 (1969) 43-52 4. Dikken, H. and E. Kmety: Serological typing methods of leptospiras. In: Methods in Microbiology, vol. 11, pp. 259-308 (T. Bergan and]. R. Norris, eds.). Academic Press, London (1978) 5. Ey, P. 1., S. J. Prowse, and C. R. Jenkin: Isolation of pure IgGl, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry 15 (1978) 429-436 6. Kida, H., 1. E. Brown, and R. G. Webster: Biological activity of monoclonal antibodies to operationally defined antigenic regions on the hemagglutinin molecule of Alseal/ Massachusetts/1/80(H7N7) influenza virus. Virology 122 (1982) 38-47 7. Kobayashi, Y., T. Tamai, T. Oyama, H. Hasegawa, T. Shiroguchi, E. Sada, T. Kusaba, and M. Hamaji: Identification of etiologicalleptospires of Weil's disease using monoclonal antibodies. Kansenshogaku Zasshi 57 (1983) 846-852 (in Japanese) 8. Mekler, 1. B., S. M. Klimenko, G. E. Dobrezou, V. K. Naumoua, Y. P. Hoffman, and V. M. Zhdanov: Cytochemical and immunochemical analysis at the electron microscopy level: obtaining contrasting antibodies by use of iodine. Nature (Lond.) 203 (1964) 717-719 9. Ono, E., M. Naiki, and R. Yanagawa: Production and characterization of monoclonal antibodies to lipopolysaccharide antigen of Leptospira interrogans serovar kremastos and canicola. Zbl. Bakt. Hyg., I. Abt, Orig. A 252 (1982) 414-424 10. Osechinsky, I. V., 1. B. Mekler, R. V. Petrou, and V. M. Mityushin: The use of iodinated antibodies for the location of antigens on the surface of Leptospira canicola by electron microscopy. Immunology 16 (1969) 427-431 11. Sbenberg, E.: Growth of pathogenic Leptospira in chemically defined media. J. Bact. 93 (1967) 1598-1606 12. Yanagawa, R. and 1. Takashima: Leptospira serotype kremastos of bovine origin. Jpn. J. Microbiol. 16 (1972) 147-148
Professor Dr. Ryo Yanagauia, Dept. of Hygiene and Microbiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan
Production of Monoclonal Antibodies Against Leptospiral Genus-Specific Protein Antigen and Localization of the Antigen by Immunoe1ectron Microscopy NORITSUGU SAKAMOTO, ETSURO ONO, HIROSHI KIDA, and RYO YANAGAWA Department of Hygiene and Microbiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan (Head: Prof. Dr. Yanagawa) With 2 Figures' Received January 14, 1985 . Accepted February 2, 1985
Abstract Monoclonal antibodies were first produced against a leptospiral genus-specific protein antigen (GP-Ag) isolated from Leptospira interrogans serovar kremastos strain Kyoto. The representative monoclonal antibody (GP-?) did not agglutinate kremastos Kyoto but reacted with Triton X-100 treated kreamastos Kyoto, andamana, patoc and Urawa in enzyme-linked immunosorbent assay (ELISA), indicating that the monoclonal antibody recognized the genus-specific determinant of GP-Ag. The localization of GP-Ag was investigated by immunoelectron microscopy with the iodinated antibody and peroxidase-labeled antibody. The genus-specific determinant of GPAg recognized by GP-? was located on the subsurface of the leptospiral envelope. Zusammenfassung Monoklonale Antikorper wurden zunachsr gegen ein genus-spezifisches Protein-Antigen (GP-Ag) gebildet, das aus Leptospira interrogans, serovar kremastos, Stamm Kyoto isoliert worden war. Der reprasentative monoklonale Antikorper (GP-?) agglutinierte kremastos Kyoto nicht, reagierte aber mit Triton-X-lOO-behandeiten kremastos Kyoto, andamana, patoc und Urawa im ELISA, was anzeigt, daf der monoklonale Antikorper die genusspezifische Determinante von GP-Ag erkannte. Die Lokalisation von GP-Ag wurde immunelektronenmikroskopisch mit jodierten Antikorpern und peroxidasemarkierten Antikorpern untersucht. Die durch GP-? erkannte genus-spezifische Determinante von GP-Ag war unterhalb der Oberflache der BakterienhiilIe lokalisiert.
Introduction Monoclonal antibodies against leptospiral antigens have been produced by Ono et a1. (9) and Kobayashi et a1. (7) against serovar-specific antigens, and by Adler and Paine (2) against a species-specific antigen. But up to now, no monoclonal antibodies have been produced against leptospiral genus-specific antigen.
558
N. Sakamoto, E. Ono, H. Kida, and R. Yanagawa
A partially purified leptospiral genus-specific protein antigen (GP-Ag) was isolated from Leptospira interrogans serovar kremastos strain Kyoto by treating the organisms with Triton X-lOa followed by using a purification procedure including fractionation with DEAE-cellulose column chromatography and ethanol precipitation. The molecular weight of GP-Ag was estimated to be approximately 62 000. The protein part of GP-Ag which was responsible for its genus-specificity did not appear to be located on the leptospiral cell surface, because the antiserum to GP-Ag did not agglutinate L. biflexa and the serovars of L. interrogans except those belonging to Hebdomadis serogroup (In press). Little information is available on the localization of leptospiral antigens by immunoelectron microscopy. Only one report was published by Osechinsky et al. (10), who showed that iodinated mouse anti-canicola antibodies bound to the surface of the homologous leptospiras. In the present study, a monoclonal antibody against GP-Ag was produced and the localization of GP-Ag was investigated by immunoelectron microscopy with the monoclonal antibody. Materials and Methods Production of monoclonal antibody to the leptospiral genus-specific protein antigen
GP-Ag was purified from Leptospira interrogans serovar kremastos strain Kyoto (12) grown in Shenberg's synthetic medium (11) according to Sakamoto et al. (In press). Monoclonal antibody to GP-Ag was prepared in mouse hybridorna system (6). The spleen cell donor was a BALB/c mouse subcutaneousl y and intramuscularly immunized with 0.25 ml of emulsion consisting of one part of phosphate-buffered saline (PBS, pH 7.2) containing 500 I!gGP-Ag, and one part of Freund's complete adjuvant three times at 7-day intervals. Fusion was carried out 2 months later, 3 days after an intraperitoneal booster injection of 500 ug of GP-Ag. Monoclonal antibody to the serovar-specific antigen
Monoclonal antibody (KT-1) to the serovar-specific antigen (TM antigen) of kremastos strain Kyoto produced by Ono et al. (9) was used. Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed virtually according to the method described (6). Triton X-lOOtreated kremastos Kyoto, andamana, patoc and Urawa were used as coating antigen. Microscopic agglutination test
Microscopic agglutination test was done as reported (4). Iodinated antibody and horseradish peroxidase-labeled antibody
IgG-fractions from mouse ascitic fluids purified by the method of Ey et al. (5) were iodinated by the method of Mekler et al. (8) and labeled with horseradish peroxidase (Sigma Chern. Co., St. Louis, USA; EC 1.11.1.7) by the method of Avrameas (3). Immunoelectron microscopic technique
Immunoelectron microscopic analysis using iodinated monoclonal antibody was done according to Mekler et al. (8). Samples were examined under a Hitachi HU-12A electron microscope.
Localization of Leptospiral Genus-Specific Antigen
559
Immunoelectron microscopic analysis using horseradish peroxidase (HRPO)-labeled antibody was done as follows. Leptospiral cells in 10 ml of Shenberg's synthetic medium were incubated for 3 h at room temperature with 100 III of the HRPO-Iabeled antibody preparat ions. A hundred III of substrate solution (0.05 M Tris-HCl buffer, pH 7.6, 6 mM 3.3'-diaminobenzidine-tetrahydrochloride, 0.05 %, vlv, hydrogen peroxide) were added, and this solution was incubated for 30 min at room temperature. The material was fixed in OS04, dehydrated and embedded in Epon. Ultrathin sections were examined under a Hitachi HU-12A electron microscope. Results
Reactivities of mon oclonal antibodies to GP-Ag Four hybridoma cell lines were established against GP-Ag. Of these, GP-7 did not agglut inate the homologous kremastos strain Kyoto in microscopic agglutination test (negative at 1: 10) (Ta ble 1). GP-7 reacted with kremastos Kyoto of L. interrogans, and andaman a, patoc and Urawa of L. biflexa in ELISA. The remaining 3 antibodies gave similar results. KT-1, which was a monoclonal antibody to TM antigen , agglutinated kremastos in microscopi c agglutination test, and reacted with kremastos but not with patoc in ELISA.
lmmunoelectron microscopy with iodinated monoclonal antibody No iodin ated GP-7 was bound to the lepto spiral cell (Fig. 1a). On the other hand, iodinated KT-1 was bound to the surface of the lepto spiral cell (Fig. 1b). Thu s, it was consid ered that the antigenic determin ant of GP-Ag recognized by GP-7 was not located on the surface of the lepto spiral envelope, while the ant igenic determin ant of the TM anti gen recognized by KT-I was located on the leptospiral cell surface. Figure l c shows control organisms with no added iodinated antibody.
lmmunoeleetron microscopy with HR PO-Iabeled monoclonal antib ody No labeled GP-7 was bound to the lepto spiral cell (Fig. 2a). Labeled GP-7 was bound to the lepto spiral envelope only when the lepto spiral cells were dam aged and the envelope was released from its protoplasmic cylinder (Fig. 2b). On the other hand, labeled KT-1 was bound to the surface of the leptospiral cell (Fig. 2c). Figure 2d shows control organisms with no add ed HRPO-I abeled antib ody. Thus, it was considered that the ant igenic determin ant of GP-Ag recognized by GP-7 was located on the sub surface of the lepto spiral envelope, while th e antigenic determ inant of the TM antigen recognized by KT-1 was located on the leptospiral cell surface. Discussion Mon oclon al antibodies to the leptospiral genus-specific protein antigen (GP-Ag) were produced for the first time. The monocl onal antib odies, including GP-7, did not agglutinate kremastos Kyoto but reacted with Triton X-lO O-treated kremastos Kyoto , andam ana, patoc and Urawa in ELISA. The localization of GP-Ag by immunoelectron microscopy with GP-7 show ed that the antigenic determ inant of GP-Ag recognized by GP-7 was located on the subsurface of the leptospiral envelope . This finding does not contradict the fact th at the antiserum
102,400 409,600
< 10
128,000
ELISA wirlr' kremastos
MAT with 2 kremastos 12,800 NT 4
ELISA with andamana 102,400
< 100
ELISA with patoc
25,600 NT
ELISA with Urawa
< 100 NT
ELISA with Leptonema illini
Immunoglobulin was tested for the class of immunoglobulin light and heavy chains by double immunodiffusion using specific antisera to various kinds of myeloma protein or each light chain subclass (Miles Lab., Inc., Elkhart, Ind., USA) . Reciprocal of the dilution of monoclonal antibo dies expressed in 50% microscopic agglutination. MAT: microscopic agglutination test. Titers are expressed as the highest dilution of antibody to shows significant binding above background level. NT: not tested .
yl. x. y3. x.
GP-7 KT-1
1
Irnmunoglobulin 1 type
Monoclonal antibody
Table 1. Reactivities of monoclonal antibodies to GP-Ag (GP-7) and TM antigen (KT-1)
"" 0'\
I>l
~
OQ
I>l
::s
~
?:l
0-
~ 0J' I>l ::s
;:r::
.0
oe
.'0 rr.'
o
3
I>l
;.;-
I>l
en
;z:
o
Localization of Leptospiral Genus-Specific Antigen
561
a
b
c
Fig. 1. Electronmicrographs of L. interrogans serovar kremastos strain Kyoto added with iodinated monoclonal antibody GP-7 (a), iodinated monoclonal antibody KT-1 (b), and with no added iodinated antibody (c) (x 25,000 ). 34 Zbl. Bakt. Hyg. A 25914
562
N. Sakamoto , E.Ono, H. Kida, and R. Yanagawa
.c
"
o Fig. 2. Electronmicrograph s of ultrathinsection of L. interrogans serovar kremastos strain Kyoto added with HRPO -labeled monoclonal antibody GP-? (a and b), HRPO-Iabeled monoclonal antibody KT-l (e), and with no added HRPO-Iabeled antibody (d) (x 60,000 ).
Localization of Leptospiral Genus-Specific Antigen
563
to GP-Ag did not agglutinate 1. biflexa and the serovars with the exceptions of those belonging to Hebdomadis serogroup of 1. interrogans (In press). Results of immunoelectron microscopic analysis of localization of serovar-specific (TM) antigen showed that TM antigen was located on the leptospiral cell surface. This finding agrees with the fact that TM antigen was associated with microscopic agglutination and that TM antigen inhibited leptospiral agglutination (1). The location on the subsurface of GP-Ag in the leptospiral envelope may lead to further studies on the role of GP-Ag in the architecture of the leptospiral envelope and on function of GP-Ag such as transportion of low molecular substances, etc. The use of monoclonal antibody against GP-Ag may be helpful not only in elucidating localization of GP-Ag, but also in isolating and characterizing the antigenic determinant of GP-Ag, which will be the subject of a future study.
References 1. Adachi, Y. and R. Yanagawa: Inhibition of leptospiral agglutination by the type-specific main antigens of leptospiras. Infect. Immun. 17 (1977) 466-467 2. Adler, B. and S. Paine: Species- and genus-specific antigens in leptospira, revealed by monoclonal antibodies and enzyme immunoassay. Zbl. Bakt. Hyg., I. Abt. Orig. A 255 (1983) 317-322 3. Avrameas, S.: Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry 6 (1969) 43-52 4. Dikken, H. and E. Kmety: Serological typing methods of leptospiras. In: Methods in Microbiology, vol. 11, pp. 259-308 (T. Bergan and]. R. Norris, eds.). Academic Press, London (1978) 5. Ey, P. 1., S. J. Prowse, and C. R. Jenkin: Isolation of pure IgGl, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry 15 (1978) 429-436 6. Kida, H., 1. E. Brown, and R. G. Webster: Biological activity of monoclonal antibodies to operationally defined antigenic regions on the hemagglutinin molecule of Alseal/ Massachusetts/1/80(H7N7) influenza virus. Virology 122 (1982) 38-47 7. Kobayashi, Y., T. Tamai, T. Oyama, H. Hasegawa, T. Shiroguchi, E. Sada, T. Kusaba, and M. Hamaji: Identification of etiologicalleptospires of Weil's disease using monoclonal antibodies. Kansenshogaku Zasshi 57 (1983) 846-852 (in Japanese) 8. Mekler, 1. B., S. M. Klimenko, G. E. Dobrezou, V. K. Naumoua, Y. P. Hoffman, and V. M. Zhdanov: Cytochemical and immunochemical analysis at the electron microscopy level: obtaining contrasting antibodies by use of iodine. Nature (Lond.) 203 (1964) 717-719 9. Ono, E., M. Naiki, and R. Yanagawa: Production and characterization of monoclonal antibodies to lipopolysaccharide antigen of Leptospira interrogans serovar kremastos and canicola. Zbl. Bakt. Hyg., I. Abt, Orig. A 252 (1982) 414-424 10. Osechinsky, I. V., 1. B. Mekler, R. V. Petrou, and V. M. Mityushin: The use of iodinated antibodies for the location of antigens on the surface of Leptospira canicola by electron microscopy. Immunology 16 (1969) 427-431 11. Sbenberg, E.: Growth of pathogenic Leptospira in chemically defined media. J. Bact. 93 (1967) 1598-1606 12. Yanagawa, R. and 1. Takashima: Leptospira serotype kremastos of bovine origin. Jpn. J. Microbiol. 16 (1972) 147-148
Professor Dr. Ryo Yanagauia, Dept. of Hygiene and Microbiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan