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Establishment of hybridomas secreting monoclonal antibodies to Chlamydia psittaci
Immunology Letters, 10 (1985) 325-327 Elsevier
Imlet 641
ESTABLISHMENT OF HYBRIDOMAS SECRETING MONOCLONAL ANTIBODIES TO CHLAMYDIA PSITTACI V. FUENTES 1, J. F. LEFEBVRE 1, E LEMA 2, E. BISSAC I and J. ORFILA 1 1Laboratoire de Bact~riologie-Immunologie G~ndrale, Facultd de Mddecine d'Amiens, 12, rue E Petit, 80036 Amiens and 2Laboratoire d'Immunologie Cellulaire, Institut Pasteur 25-28, rue du Dr Roux, 75724 Paris Cedex 15, France
(Received 1 March 1985) (Accepted 16 May 1985)
1. Summary
3. Materials and Methods
Eleven stable anti-Chlamydia hybrid clones by fusions between X63-Ag8653 myelomas and immune splenocytes from Chlamydia psittaci immunized F1 (C57BL6xBALB/c) mice have been established which react with the 12 reference Chlamydia strains (seven C. trachomatis and five C. psittaci. Ten of these monoclonal antibodies are directed against the genera-specific epitope (40,000 MW component) for which prolonged immunization seems to be responsible.
3.1. Chlamydia strain
2. Introduction The application of monoclonal antibody immuno-diagnostic tools for an improved rapid diagnosis of Chlamydia psittaci infection is important since these agents are responsible for a variety of human diseases. Recently, Stephens et al. [1] produced monoclonal antibodies against Chlamydia trachomatis species. In the present studies, monoclonal antibodies have been produced against C. psittaci aimed at production of monoclonals against genus-specific antigens besides their application to rapid diagnosis of chlamydiosis in man and animals.
Key words." Chlamydia psittaci - monoclonal antibodies
Chlamydia psittaci (Loth strain) grown in L 929 cells and purified on renografin gradient [2] was employed. For immunoblotting studies, chlamydial outer membrane complex (COMC) containing major outer protein (MOP) was isolated by sarkosyl extraction method [3]. 3.2. Mice F1 hybrids (C57BL6XBALB/c) were used for immunization as these mice are not lethally susceptible to C. psittaci, unlike BALB/c, and are high humoral immune responders. 3.3. Immunization F1 mice (C57BL6×BALB/c) were immunized intraperitoneally with purified Chlamydia (10 LD100) and boosted after 6 weeks with the same dose of organisms. The spleens were removed aseptically 3 days later for somatic hybridization.
3.4. Cell fusion X63-Ag8653 myeloma cells were fused with immune splenocytes using PEG 4000 with the standard procedure [4]. Stable hybrids were selected in HAT (hypoxanthine, aminopterin and thymidine) medium and further cloned. 3.5. Screening for anti-Chlamydia antibodies The micro-immunofluorescence (micro-IF) technique of Wang and Grayston [5] was used to
0165-2478 / 85 / $ 3.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
325
detect Chlamydia-specific monoclonal antibodies employing C. trachomatis (LB1 strain) and C. psittaci (Loth strain) as reference strains. Specific anti-Chlamydia antibody-producing hybrids were expanded and grown in F1 mice (C57B16 × BALB/c) as ascites. 3.6. Characterization of monoclonal antibodies The isotypicity of monoclonal antibodies was determined by a double immunodiffusion against anti-mouse 3'1, 72a, 72b, 3,3 and/~ antisera (provided by Cappel). The specificity of these antibodies was assessed by micro-IF on a panel of 12 classified referenced Chlamydia strains provided by Dr E Eb. The strains included seven C. trachomatis immunotypes (A/SA 1, D/LB 1, E/D/220, H/UW4, J/VW36, L1/44 OL and L3/404L, and five C. psittaci immunotypes described previously [6]: 1/6 BC (psittacosis), 2/ewes abortion, 3/LW 679, (ovine polyarthritis), 4FP/145 (feline pneumonitis and 1 A/MP1 (murine meningopneumonitis). The immunofluorescent assays were also performed on L929 cells infected with LB1 and Loth strains. 3.7. lmmunoblotting technique COMC-purified preparations were dissolved in sample buffer (2°7o sodium dodecylsulfate, 5% 2-mercapto-ethanol, 0.001% bromophenol blue, 10070 glycerol in 0.06 M Tris-HCl, pH 6.8) at 100°C for 3 min. The samples were electrophoresed under reducing conditions in 10% polyacrylamide slab gels [7]. The proteins were then electrophoretically transferred to a nitrocellulose sheet at 5 V/cm for 30 min in 6 M urea. The membrane was incubated in phosphatebuffered saline (PBS) containing 10% fetal calf serum for 30 min at 22°C, washed with three changes of PBS, and further incubated in undiluted hybridome culture supernatants for 2 h at 37°C. After washing with PBS as described earlier, the immune complexes were detected by anti-mouse Ig coupled to horse radish peroxidase (IPP, Paris) with 3,3'-diamino-benzidine as substrate.
326
Table 1 Summary of results Antibodies
Heavy chain (isotype)
Blotting (M.W.)
Specificity
AD 5 DA 7 DH 2 AE 8 BC 10 BG 4 DB 6 EE 2 DF 10 BD 2 EB 11
/x 3,2a 72a 3'2a 3,3 3,2a ND 3,3 3,3 3,2a 3,2a
40,000 40,000 ND ND 40,000 ND ND 40,000 ND 40,000 ND
genus genus genus genus genus genus genus genus genus genus type
4. Results and Discussion
The results are summarized in Table 1. Two major observations can be made. (A) Eleven stable anti-Chlamydia hybrid clones have been obtained which reacted with all the twelve tested Chlamydia strains. One hybrid (EBll) was specific for immunotype 1 while antibodies from three other hybrids (EE2, DB6 and AE6) reacted with several strains of both the Chlamydia species but not with any of the aforesaid twelve strains. The prolonged immunization seems to be responsible for such disproportion between genera- and type-specific antibodies. (B) Some of the monoclonal antibodies that showed genus specificity by micro-IF, reacted in COMC immunoblots with a 40,000 MW component (probably, MOP). Stephens et al. [1] have previously demonstrated that MOP is a speciesspecific antigen. It is hypothesized that C. trachomatis MOP has two epitopes, one of which is genus-specific, the other being speciesspecific. The monoclonal antibodies to C. trachomatis produced by BALB/c immune spleen cells and NS1 myelomas fusion recognize the speciesspecific epitope but not the genera-specific one. Most monoclonal antibodies (10 out of 11 hybridoclones) to C. psittaci produced by fusion between F1 mice (C57BL6 ×BALB/c) immune splenocytes and X62Ag8653 myelomas recognize the genus-specific epitope.
References [1] Stephens, R., Tam, M., Kuo, C. C. and Nowinsky, R. (1982) J. Immunol. 128, 1083-1089. [2] Kuo, C. C., Wang, S. P. and Grayston, J. T. (1977) in: Nongonococcal Urethritis and Related Infections (Hobson, D. and Holmes, K. K., Eds.) p. 328, American Society for Microbiology, Washington. [3] Caldwell, H., Kromhout, J. and Schachter, J. (1981) Infect. Immun. 31, 1161-1176.
[4] Kohler, G. and Milstein, C. (1975) Nature (London) 256, 495 - 497. [5] Wang, S. P. and Grayston, J. T. (1970) Am. J. Ophthalmol. 70, 367-374. [6] Eb, E and Orfila, J. (1982) Infect. Immun. 37, 1289-1291. [7] Laemmli, U. K. (1970) Nature (London) 227, 680-685.
327
Imlet 641
ESTABLISHMENT OF HYBRIDOMAS SECRETING MONOCLONAL ANTIBODIES TO CHLAMYDIA PSITTACI V. FUENTES 1, J. F. LEFEBVRE 1, E LEMA 2, E. BISSAC I and J. ORFILA 1 1Laboratoire de Bact~riologie-Immunologie G~ndrale, Facultd de Mddecine d'Amiens, 12, rue E Petit, 80036 Amiens and 2Laboratoire d'Immunologie Cellulaire, Institut Pasteur 25-28, rue du Dr Roux, 75724 Paris Cedex 15, France
(Received 1 March 1985) (Accepted 16 May 1985)
1. Summary
3. Materials and Methods
Eleven stable anti-Chlamydia hybrid clones by fusions between X63-Ag8653 myelomas and immune splenocytes from Chlamydia psittaci immunized F1 (C57BL6xBALB/c) mice have been established which react with the 12 reference Chlamydia strains (seven C. trachomatis and five C. psittaci. Ten of these monoclonal antibodies are directed against the genera-specific epitope (40,000 MW component) for which prolonged immunization seems to be responsible.
3.1. Chlamydia strain
2. Introduction The application of monoclonal antibody immuno-diagnostic tools for an improved rapid diagnosis of Chlamydia psittaci infection is important since these agents are responsible for a variety of human diseases. Recently, Stephens et al. [1] produced monoclonal antibodies against Chlamydia trachomatis species. In the present studies, monoclonal antibodies have been produced against C. psittaci aimed at production of monoclonals against genus-specific antigens besides their application to rapid diagnosis of chlamydiosis in man and animals.
Key words." Chlamydia psittaci - monoclonal antibodies
Chlamydia psittaci (Loth strain) grown in L 929 cells and purified on renografin gradient [2] was employed. For immunoblotting studies, chlamydial outer membrane complex (COMC) containing major outer protein (MOP) was isolated by sarkosyl extraction method [3]. 3.2. Mice F1 hybrids (C57BL6XBALB/c) were used for immunization as these mice are not lethally susceptible to C. psittaci, unlike BALB/c, and are high humoral immune responders. 3.3. Immunization F1 mice (C57BL6×BALB/c) were immunized intraperitoneally with purified Chlamydia (10 LD100) and boosted after 6 weeks with the same dose of organisms. The spleens were removed aseptically 3 days later for somatic hybridization.
3.4. Cell fusion X63-Ag8653 myeloma cells were fused with immune splenocytes using PEG 4000 with the standard procedure [4]. Stable hybrids were selected in HAT (hypoxanthine, aminopterin and thymidine) medium and further cloned. 3.5. Screening for anti-Chlamydia antibodies The micro-immunofluorescence (micro-IF) technique of Wang and Grayston [5] was used to
0165-2478 / 85 / $ 3.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
325
detect Chlamydia-specific monoclonal antibodies employing C. trachomatis (LB1 strain) and C. psittaci (Loth strain) as reference strains. Specific anti-Chlamydia antibody-producing hybrids were expanded and grown in F1 mice (C57B16 × BALB/c) as ascites. 3.6. Characterization of monoclonal antibodies The isotypicity of monoclonal antibodies was determined by a double immunodiffusion against anti-mouse 3'1, 72a, 72b, 3,3 and/~ antisera (provided by Cappel). The specificity of these antibodies was assessed by micro-IF on a panel of 12 classified referenced Chlamydia strains provided by Dr E Eb. The strains included seven C. trachomatis immunotypes (A/SA 1, D/LB 1, E/D/220, H/UW4, J/VW36, L1/44 OL and L3/404L, and five C. psittaci immunotypes described previously [6]: 1/6 BC (psittacosis), 2/ewes abortion, 3/LW 679, (ovine polyarthritis), 4FP/145 (feline pneumonitis and 1 A/MP1 (murine meningopneumonitis). The immunofluorescent assays were also performed on L929 cells infected with LB1 and Loth strains. 3.7. lmmunoblotting technique COMC-purified preparations were dissolved in sample buffer (2°7o sodium dodecylsulfate, 5% 2-mercapto-ethanol, 0.001% bromophenol blue, 10070 glycerol in 0.06 M Tris-HCl, pH 6.8) at 100°C for 3 min. The samples were electrophoresed under reducing conditions in 10% polyacrylamide slab gels [7]. The proteins were then electrophoretically transferred to a nitrocellulose sheet at 5 V/cm for 30 min in 6 M urea. The membrane was incubated in phosphatebuffered saline (PBS) containing 10% fetal calf serum for 30 min at 22°C, washed with three changes of PBS, and further incubated in undiluted hybridome culture supernatants for 2 h at 37°C. After washing with PBS as described earlier, the immune complexes were detected by anti-mouse Ig coupled to horse radish peroxidase (IPP, Paris) with 3,3'-diamino-benzidine as substrate.
326
Table 1 Summary of results Antibodies
Heavy chain (isotype)
Blotting (M.W.)
Specificity
AD 5 DA 7 DH 2 AE 8 BC 10 BG 4 DB 6 EE 2 DF 10 BD 2 EB 11
/x 3,2a 72a 3'2a 3,3 3,2a ND 3,3 3,3 3,2a 3,2a
40,000 40,000 ND ND 40,000 ND ND 40,000 ND 40,000 ND
genus genus genus genus genus genus genus genus genus genus type
4. Results and Discussion
The results are summarized in Table 1. Two major observations can be made. (A) Eleven stable anti-Chlamydia hybrid clones have been obtained which reacted with all the twelve tested Chlamydia strains. One hybrid (EBll) was specific for immunotype 1 while antibodies from three other hybrids (EE2, DB6 and AE6) reacted with several strains of both the Chlamydia species but not with any of the aforesaid twelve strains. The prolonged immunization seems to be responsible for such disproportion between genera- and type-specific antibodies. (B) Some of the monoclonal antibodies that showed genus specificity by micro-IF, reacted in COMC immunoblots with a 40,000 MW component (probably, MOP). Stephens et al. [1] have previously demonstrated that MOP is a speciesspecific antigen. It is hypothesized that C. trachomatis MOP has two epitopes, one of which is genus-specific, the other being speciesspecific. The monoclonal antibodies to C. trachomatis produced by BALB/c immune spleen cells and NS1 myelomas fusion recognize the speciesspecific epitope but not the genera-specific one. Most monoclonal antibodies (10 out of 11 hybridoclones) to C. psittaci produced by fusion between F1 mice (C57BL6 ×BALB/c) immune splenocytes and X62Ag8653 myelomas recognize the genus-specific epitope.
References [1] Stephens, R., Tam, M., Kuo, C. C. and Nowinsky, R. (1982) J. Immunol. 128, 1083-1089. [2] Kuo, C. C., Wang, S. P. and Grayston, J. T. (1977) in: Nongonococcal Urethritis and Related Infections (Hobson, D. and Holmes, K. K., Eds.) p. 328, American Society for Microbiology, Washington. [3] Caldwell, H., Kromhout, J. and Schachter, J. (1981) Infect. Immun. 31, 1161-1176.
[4] Kohler, G. and Milstein, C. (1975) Nature (London) 256, 495 - 497. [5] Wang, S. P. and Grayston, J. T. (1970) Am. J. Ophthalmol. 70, 367-374. [6] Eb, E and Orfila, J. (1982) Infect. Immun. 37, 1289-1291. [7] Laemmli, U. K. (1970) Nature (London) 227, 680-685.
327