Jun 5, 1984 - aminopterin-thymidine as the selection medium. After 2 weeks, the culture supernatants were tested for antibody reactivity against B. gingivalis ...
INFECTION AND IMMUNITY, OCt. 1984, p. 285-287
Vol. 46, No. 1
0019-9567/84/100285-03$02.00/0 Copyright C 1984, American Society for Microbiology
Production of Monoclonal Antibodies That Recognize Specific and Cross-Reactive Antigens of Bacteroides gingivalis SHIGEMASA HANAZAWA,* KIEKO SAITOH, YOSHIHIRO OHMORI, HIROJI NISHIHARA, SHINICHI FUJIWARA, AND SHIGEO KITANO Department of Oral Microbiology, Josai Dental University, Keyakidai, Sakado City, Saitama, 350-02, Japan Received 14 March 1984/Accepted
5
June 1984
Four monoclonal antibodies directed against Bacteroides gingivalis were established by hybridoma technoloTheir reactivity against B. gingivalis, Bacteroides intermedius, and Bacteroides melaninogenicus was detected by the enzyme-linked immunosorbent assay. Three monoclonal antibodies specifically reacted with B. gingivalis. One recognized antigens that were cross-reactive between B. gingivalis and B. intermedius. These monoclonal antibodies provide new tools for antigenic analysis of B. gingivalis. gy.
Recent studies have suggested that human periodontal disease is closely associated with gram-negative anaerobic oral bacteria (2, 11, 12). In this regard, Bacteroides gingivalis has been isolated in relatively high proportions from inflamed gingival pockets of patients with adult periodontal disease. Some investigators indicated that immunoglobulin G (IgG) serum antibodies against B. gingivalis were increased significantly in patients with adult periodontal disease (5, 7). Therefore, B. gingivalis is now believed to be associated with the pathogenesis of this disease. Reed et al. (10) suggested that B. gingivalis does not share major antigens with nonoral Bacteroides asaccharolyticus or with oral and nonoral Bacteroides melaninogenicus subsp. However, antigenic analysis of B. gingivalis was not investigated in detail. Clearly, for a better understanding of the host immune response against B. gingivalis more information about the characteristics of specific antigens of B. gingivalis is necessary. Hybridization technology (3) has made it possible to use monoclonal antibodies as immunological probes. As far as we know, monoclonal antibodies against specific and crossreactive antigens of B. gingivalis have not been previously obtained. We report here the production and partial characterization of monoclonal antibodies prepared by the fusion of SP2/OAg/4 myeloma cells to spleen cells from BALB/c mice immunized with B. gingivalis 381. The strains used in this study are shown in Table 1. All strains were cultivated with EX-1 diffusate medium (6) for 48 h at 37°C under anaerobic conditions. The cells were then washed and lyophilized. Formalin-treated B. gingivalis 381 (1 mg [dry weight] per mouse) was injected intraperioneally into BALB/c mice. Boosters with the same bacterium and dosage were given intravenously 4 days before harvesting the spleens. Hybridomas were established according to the protocol of Oi and Herzenberg (9). Briefly, the washed, sensitized spleen cells were combined with SP2/0 myeloma cells at a ratio of 5 to 1. The cell mixture was pelleted, washed, and then fused with 1 ml of 42.5% polyethylene glycol 4000 (E. Merck AG). After fusion, the cell suspension was washed and suspended in RPMI 1640 medium containing 10% fetal bovine serum (Flow Laboratories). The suspended cells were inoculated at 2.5 x 105 cells per well into Falcon microculture plates *
(3040). The cultures were fed RPMI 1640-hypoxanthineaminopterin-thymidine as the selection medium. After 2 weeks, the culture supernatants were tested for antibody reactivity against B. gingivalis 381 by the enzyme-linked immunosorbent assay (ELISA) (1) with alkaline phosphatase-conjugated rabbit anti-mouse immunoglobulin. Positive hybridomas were cloned by limiting dilution with thymus cells as feeder cells. The clones producing high-titer antibody were chosen for further characterization. They were grown in large quantities in vitro and injected into the peritoneums of pristene-primed BALB/c mice, and then ascites fluids were harvested. The specificity of the monoclonal antibodies was evaluated by the ELISA with sonicated extracts of B. gingivalis, Bacteroides intermedius, B. melaninogenicus, and nonoral B. asaccarolyticus. Of 120 wells, 98 showed growth of hybrid cells after selection in RPMI 1640-hypoxanthine-aminopterin-thymidine medium. Culture supernatants from 98 wells were tested, and of these only 14 reacted with the immunizing strain, B. gingivalis 381. Four clones were established by limiting dilution, and their antibodies were designated as BGF7, BGF2, BGD2, and BGE2. In double-immunodiffusion experiments by the Ouchterlony technique, monoclonal antibodies BGF7 and BGF2 were identified as IgG2a, and BGD2 and BGE2 were identified as IgG3. Table 1 shows the reactivity of the monoclonal antibodies with B. gingivalis, B. intermedius, B. melaninogenicus, and nonoral B. asaccharolyticus. All monoclonal antibodies strongly reacted against B. gingivalis but did not react with B. melaninogenicus and nonoral B. asaccharolyticus. Only BGF2 monoclonal antibody also reacted with B. intermedius (strains 20.3, JP, and ATCC 25261). Monoclonal antibody BGF2 was further tested for a comparison of its immunoreactivity against B. gingivalis 381, B. intermedius 20.3, B. melaninogenicus ATCC 15930, and nonoral B. asaccharolyticus ATCC 25260. It reacted against B. intermedius 20.3, with ca. 80% of the absorbance found for B. gingivalis 381, when the antibody was used at dilutions of 10-2 and 10-3 (Fig. 1). The above results were further extended by adsorption experiments. Each bacterial strain (5 mg [dry weight]) was incubated with 1 ml of a 10-2 dilution of monoclonal antibody (BGF2, BGE2). After incubation for 3 h at 37°C, the remaining activity of the adsorbed antibodies were subsequently tested on B. gingivalis 381. The results are shown in Fig. 2. Monoclonal antibody BGE2 was clearly
Corresponding author. 285
286
INFECT. IMMUN.
NOTES
TABLE 1. ELISA immunoreactivity of monoclonal antibodies against various strains of B. gingivalis and B. melaninogenicus subsp.' Monoclonal antibodyb (immunoglobulin) BGD2 BGF7 BGF2 BGE2 (IgG3) (IgG2a) (IgG2a) (IgG3)
Organism and strain
B. gingivalis 381 JG.1
+4 +4
+4 +4
+4 +4
+4 +4
B. intermedius 20.3 JP ATCC 25261
0 0 0
+3 +3 +1
0 0 0
0 0 0
B. melaninogenicus ATCC 15930 1.13
0 0
0 0
0 0
0 0
B. asaccharolyticus ATCC 25260
0
0
0
0
0r2
zov >
tjjL.0
O.D. 405nm OA
0.6
0.8
1.0
1.2
None 381.. 2 5 2 61 .................................... 2 0.3 1.1(3 15930
..................
..........................................
(a) BGE2 .......
...................................................................
252 6C
None 1111111111110 381
a Immunoreactivity was determined by ELISA with bacterial sonicated extracts (protein concentration, 10 jig) coated on Falcon microassay plates. b Monoclonal antibodies were obtained from ascitic fluid and then used at a dilution of lo-3. The color development was scored visually from 0 to +4.
adsorbed with B. gingivalis 381 but not with other Bacteroides species. On the other hand, monoclonal antibody BGF2 was quite clearly adsorbed with B. gingivalis and both B. intermedius strains. Several investigators (4, 8, 10) have shown that antiserum against B. gingivalis did not react with B. intermedius. It was
25261
20.3 = 1593 0
(b) BGF 2
1. 13 1111111111111111111111111111
25260G.lllllllllliiinlmlllll FIG. 2. Adsorption experiment with monoclonal antibodies (BGE2, BGF2) against B. gingivalis and B. melaninogenicus subsp. Monoclonal antibodies (10-2 dilution, 1 ml) from ascitic fluid were incubated with each bacterium (dry weight, 5 mg) for 3 h at 37°C. After incubation, the remaining activity of the adsorbed antibodies was tested with B. gingivalis 381 sonicated extracts (protein concentration, 10 jig) coated on Falcon microassay plates.
also suggested that the immunological specificities of both bacteria were different. However, as shown in the above results, our monoclonal antibody BGF2 reacted with both B. gingivalis and B. intermedius. Therefore, the present study indicates that at least one cross-reactive antigen is expressed between B. gingivalis and B. intermedius. We have generated some hybridoma lines which produce monoclonal antibodies reactive against specific antigens of B. gingivalis and against cross-reactive antigens of B. gingivalis and B. intermedius. These monoclonal antibodies will be further characterized via immunoblotting or other procedures to identify the antigens of B. gingivalis which are recognized by these antibodies.
1.2r, 1.0 E
r
Ad4a,nrtirvn
0.8
CD
;k
600.6
We thank I. Takazoe and K. Okuda, Department of Oral Microbiology, Tokyo Dental College, and H. Sagawa and T. Umemoto, Department of Bacteriology, Osaka Dental University for kindly supplying the bacterial strains used in this study.
0.4k
0.2 U
102
MM
-
1O'
i-M
10-4 10-5 10-3 l0- 2 AntibodY dilution FIG. 1. Comparison of immunoreactivity of monoclonal antibody BGF2 with B. gingivalis 381 (0), B. intermedius 20.3 (a), B. melaninogenicus ATCC 15930 (0), and nonoral B. asaccharolyticus ATCC 25260 (W). The reactivity was measured by ELISA with bacterial sonicated extracts (protein concentration, 10 jig) coated on Falcon microassay plates. The monoclonal antibody was obtained from ascitic fluid and then used in several dilutions.
LITERATURE CITED 1. Engvall, E., and P. Perlmann. 1972. Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzyme labeled anti-immunoglobulin in antigen-coated tubes. J.
Immunol. 109:129-135. 2. Irving, J. T., M. G. Newman, S. S. Socransky, and J. D. Herley. 1975. Histological changes in experimental periodontal disease in rats monoinfected with gram-negative organism. Arch. Oral Biol. 20:219-220. 3. Kohler, G., and C. Milstein. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 256:495-497. 4. Lambe, D. W., Jr. 1974. Determination of Bacteroides melaninogenicus serogroups by fluorescent antibody staining. Appl. Microbiol. 28:561-567. 5. Mansheim, B. J., M. L. Stenstrom, S. B. Low, and W. B. Clark.
VOL. 46, 1984
6. 7.
8. 9.
1980. Measurement of serum and salivery antibodies to the oral pathogen Bacteroides asaccharolyticus in human subjects. Arch. Oral Biol. 25:553-557. Mashimo, P. A., and S. A. Ellison. 1972. Diffusate media for cultivation of oral anaerobic bacteria. J. Dent. Health 22:38-45. Mouton, C., P. G. Hammond, J. Slots, and R. J. Genco. 1981. Serum antibodies to oral Bacteroides asaccharolyticus (Bacteroides gingivalis): relationship to age and periodontal disease. Infect. Immun. 31:182-192. Mouton, C., P. G. Hammond, J. Slots, M. J. Reed, and R. J. Genco. 1981. Identification of Bacteroides gingivalis by fluorescent antibody staining. Ann. Microbiol. (Paris) 132B:69-83. Oi, V. T., and L. A. Herzenberg. 1980. Immunoglobulin produc-
NOTES
287
ing hybrid cell lines, p. 351-371. In B. B. Mishell and S. M. Shigi (ed.), Selected methods in cellular immunology. W. H. Freeman and Co. San Francisco. 10. Reed, M. J., J. Slots, C. Mouton, and R. J. Genco. 1980. Antigenic studies of oral and nonoral black-pigmented Bacteroides strains. Infect. Immun. 29:564-574. 11. Slots, J. 1982. Importance of black-pigmented Bacteroides in human periodontal disease, p. 27-45. On R. J. Genco and S. E. Mergenhagen (ed.), Host-parasite interactions in periodontal disease. American Society for Microbiology, Washington, D.C. 12. Socransky, S. S. 1977. Microbiology of periodontal disease: present status and future consideration. J. Periodontol. 48:497504.
