INFECTION AND IMMUNITY, Aug. 1990, p. 2542-2546

Vol. 58, No. 8

0019-9567/90/082542-05$02.00/0 Copyright © 1990, American Society for Microbiology

Monoclonal Antibody against a Serotype Antigen of Porphyromonas (Bacteroides) endodontalis and Characteristics of the Antigen SHIGEMASA HANAZAWA,l* TSUYOSHI SAGIYA,2 SHIGERU AMANO,1 HIROFUMI NISHIKAWA,2 AND SHIGEO KITANO' Departments of Oral Microbiology' and Endodontics,2 Meikai University School of Dentistry, Keyakidai, Sakado City, Saitama 350-02, Japan Received 11 December 1989/Accepted 25 May 1990

Recent studies have demonstrated the presence of three serotypes (01K1, 01K2, and 01K-) of Porphyromo(Bacteroides) endodontalis. In the present study, a hybridoma cell line producing monoclonal antibody (BEEll) specific for serotype 01K, of P. endodontalis was established. The specificity of the antibody was evaluated by enzyme-linked immunosorbent assay and immunoslot blot analysis. BEEll antibody reacted with strains ATCC 35406, HG 400, and HG 421 of the bacterium. However, it did not react with HG 422 or HG 948. Also, the antibody did not react with any of the black-pigmented Bacteroides strains tested. Although the antibody reacted with total cell envelope and capsule materials, it did not do so with lipopolysaccharide. The antibody reacted with antigen material having a molecular mass of 110 kilodaltons (kDa), as judged from fractionation by Superose 12 prep gel chromatography. When the peak fraction from the Superose 12 column was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, the reactivity was detected as a single band at an apparent molecular mass of about 52 kDa. The antigen material purified partially by high-performance liquid chromatography was sensitive to trypsin, V8 protease, and heating to 80°C but not to neuraminidase. Therefore, the present study shows that BEEll antibody recognizes a serotype antigen of P. endodontalis which may be a dimer consisting of monomers having molecular masses of approximately 52 kDa and sensitivity to proteases and heat. nas

Recent studies have given rise to the concept of a narrow specific bacterial etiology of the different forms of periodontal diseases (14, 15). Certain black-pigmented Porphyromonas and Bacteroides species such as Porphyromonas (Bacteroides) gingivalis and Bacteroides intermedius play an important role in the etiology of adult periodontal disease and acute necrotizing ulcerative gingivitis. Shah and Collins (13) have recently proposed that B. gingivalis and B. endodontalis should be reclassified in a new genus, Porphyromonas, as P. gingivalis and P. endodontalis, because these black-pigmented Bacteroides spp. differ markedly in some properties from the type species of Bacteroides, B. fragilis. Several studies (3, 10, 12, 18, 19, 21) have revealed the presence of black-pigmented Bacteroides and Porphyromonas spp. in odontogenic abscesses. van Winkelhoff et al. (19) found that P. endodontalis was isolated almost exclusively from periapical abscesses of endodontal origin. They also observed that B. intermedius was the most frequently isolated species in all of the samples they examined. However, these researchers showed that the presence of this organism is not specifically correlated with periapical abscesses. Sundqvist et al. (16) also showed P. endodontalis, P. gingivalis, and B. intermedius to be prevalent in root canal infections. In light of this evidence, P. endodontalis may be an important pathogen in periapical abscesses and root canal infections. Recently, van Winkelhoff et al. (20) have suggested the presence of three serotypes (O,Kj, OjK2, and O1K-) of P. endodontalis. However, the antigenic and chemical properties of these serotypes have not yet been characterized in detail. In this study, we established a monoclonal antibody that recognizes the O,Kj serotype *

antigen of P. endodontalis, and we describe herein certain characteristics of this antigen. MATERIALS AND METHODS Bacterial strains and cultivation. P. endodontalis ATCC 35406, HG 400, HG 421, HG 422, and HG 948; P. gingivalis 381; P. asaccharolytica ATCC 25260; B. intermedius ATCC 25261 and ATCC 25611; B. melaninogenicus ATCC 25845; B. loescheii ATCC 15930; and B. corporis ATCC 33547 were used in this study. The origins, sources, and serotypes of the P. endodontalis strains used in this study are listed in Table 1. These bacterial strains were cultured in a general anaerobic medium (GAM; Nissui Pharmaceutical Co. Tokyo, Japan) supplemented with hemin (0.5 mg/ml) and menadione (5 mg/ml). These bacterial cells were harvested when they reached stationary phase and were then washed with physiological saline and lyophilized. Establishment of monoclonal antibody that recognizes a serotype antigen of P. endodontalis. Formalin-treated cells of P. endodontalis ATCC 35406 (1 mg [dry weight] per mouse) were injected intraperitoneally into BALB/c mice. Boosters with the same bacterium and at the same dosage were given after 2 weeks, and then a final booster was administered intraperitoneally 4 days before harvesting of the spleens. Hybridomas were prepared by the method of Oi and Herzenberg (11). Briefly, sensitized spleen cells were combined with SP2/0 Ag 14 myeloma cells. The cell mixture was pelleted, washed, and then fused in 1 ml of 42.5% polyethylene glycol 4000. After fusion, the cell suspension was washed and then cultured in RPMI 1640 medium containing 10% fetal bovine serum (GIBCO, Chagrin Falls, Ohio). After hypoxanthine-aminopterin-thymidine selection, the culture supernatants were tested for antibody reactivity against P. endodontalis HG 400 by enzyme-linked immunosorbent

Corresponding author. 2542

MONOCLONAL ANTIBODY AGAINST P. ENDODONTALIS ANTIGEN

VOL. 58, 1990

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TABLE 1. P. endodontalis strains used in this study Strain

Serotype

ATCC 35406 (HG 370) HG 400 HG 421 HG 422 HG 948 a ND, Not defined.

O0K1 O0K1

01K1 NDa ND

assay (ELISA) with alkaline phosphatase-conjugated goat anti-mouse immunoglobulin as described previously (2, 5). Positive hybridomas were cloned three times by limiting dilution with thymus cells as feeder cells. The clone producing the highest antibody titer was chosen. Immunoslot blot assay. Bacterial cells were sonicated for 30 min at 200 V with a sonicator (Branson Sonic Power Co., Danbury, Conn.). The sonicated extracts were centrifuged for 15 min at 12,000 x g, and the supernatants were blotted onto nitrocellulose paper in an immunoslot blot apparatus (Hybri-slot Manifold; Bethesda Research Laboratories, Gaithersburg, Md.). After blotting, the nitrocellulose paper was treated with 3% (wt/vol) gelatin and then washed with 20 mM 3Tris hydrochloride (pH 7.5) containing 0.1% (vol/vol) Tween 20 (Tris-buffered saline-Tween 20). After being washed, the paper was treated for 60 min with BEEll monoclonal antibody and subsequently washed with Trisbuffered saline-Tween 20. Antibody binding was visualized by use of horseradish peroxidase-conjugated goat antimouse immunoglobulin G (Bio-Rad Japan, Tokyo, Japan) and HRP color development reagent (Bio-Rad). SDS-PAGE. P. endodontalis sonicated extracts were boiled for 3 min with or without 5% 2-mercaptoethanol (vol/vol) in 1.25 mM Tris glycerol (pH 8.3) containing 1% (wt/vol) sodium dodecyl sulfate (SDS) and 25% (vol/vol) glycerol. The boiled materials were separated on a 10% (wt/vol) polyacrylamide gel by SDS-polyacrylamide gel electrophoresis (PAGE) according to the method of Laemmli (6). After SDS-PAGE, the proteins on the gel were visualized by silver staining. The gel was calibrated by cytochrome c monomer (molecular weight [MW], 12,400), dimer (MW, 24,800), trimer (MW, 37,200), tetramer (MW, 49,600), and hexamer (MW, 74,400). Immunoblotting. After SDS-PAGE, the separated samples were transferred to nitrocellulose paper by the method of Towbin et al. (17). After transfer, BEEll monoclonal antibody binding to the samples was visualized by use of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G and HRP color development reagent. Partial purification of serotype antigen of P. endodontalis. For the partial purification of the antigen of P. endodontalis recognized by the monoclonal antibody, strain ATCC 35406 of this species was grown anaerobically in general anaerobic medium. The harvested cells were washed with phosphatebuffered saline, suspended in the same buffer and then sonicated for 30 min at 200 V with an ultrasonicator. The sonicated extracts were concentrated with polyethylene glycol. The material (20 mg) was loaded onto a Superose 12 prep (Pharmacia Japan, Tokyo, Japan)-packed column (HR 16/50) and then eluted at a flow rate of 30 ml/h with phosphate-buffered saline. The column was calibrated with the following markers: aldolase (MW, 158,000), bovine serum albumin (MW, 68,000), chicken egg albumin (MW, 45,000), chymotrypsinogen A (MW, 25,000), and cytochrome c (MW, 12,000). The fractions (4 ml) were tested

Origin Dental root canal Oral submucosal abscess Oral submucosal abscess Oral submucosal abscess Periapical abscess

Source T. J. M. van Steenbergen T. J. M. van Steenbergen J. de Graaff J. de Graaff J. de Graaff

by ELISA for reactivity against BEEll antibody. The positive fractions were pooled, concentrated, and then dialyzed against 20 mM Tris hydrochloride (pH 8.5). The dialyzed sample (0.5 mg) was applied to a high-performance liquid chromatograph (HPLC) (LC-6A; Shimadzu, Kyoto, Japan) equipped with an IEC-DEAE Biofine column (Nihon Bunkoh, Tokyo, Japan) equilibrated with 20 mM Tris hydrochloride (pH 8.5). The flow rate was kept at 1 ml/min, and the sample was eluted with a linear gradient of NaCl from 0 to 500 mM. Fractions of 0.1 ml were collected and then tested by ELISA for reactivity against BEEll antibody. Preparation of P. endodontalis cell envelope, LPS, and capsule. The P. endodontalis ATCC 35406 cell envelope was prepared as described previously (8). The crude lipopolysaccharide (LPS) was extracted by the hot phenol-water method of Westphal and Jann (22) and then purified as described earlier (4). Also, the crude capsule was prepared by the method of Martha et al. (7). Enzymes and heat treatment. Each enzyme solution, at a final concentration of 200 ,ug/ml, was added to separate tubes containing partially purified antigen material prepared by HPLC. Trypsin (type I; Sigma Chemical Co., St. Louis, Mo.), Staphylococcus aureus V8 protease (Miles Laboratories, Inc., Elkhart, Ind.), and neuraminidase (type IV; Sigma) were used. After 10 min at 37°C, the treated antigen materials were diluted with cold coating buffer (50 mM sodium carbonate, pH 9.6, for ELISA). For the measurement of heat stability, the partially purified antigen was incubated for 10 min at selected temperatures and afterward rapidly cooled on ice. Measurement of protein and hexose. Protein and hexose were measured by the methods of Bradford (1) and Morris (9), respectively. RESULTS Production of monoclonal antibody BEEll. Culture supernatants of growing hybrid colonies were screened by ELISA for activity against the sonicated extracts of P. endodontalis HG 400. Of 12 positive hybridomas that arose, the one having the highest antibody titer was cloned three times by limiting dilution to establish a stable clone. The antibody was designated as BEEll. In double-immunodiffusion experiments by the Ouchterlony technique, BEEll antibody was identified as an immunoglobulin G2a. ELISA immunoreactivity of BEEll antibody against various strains of black-pigmented Porphyromonas and Bacteroides spp. Figure 1 shows the ELISA reactivity of BEEll antibody against P. endodontalis, P. gingivalis, B. intermedius, B. melaninogenicus, and P. asaccharolytica. BEEll antibody reacted against P. endodontalis ATCC 35406 and HG 400 but not against the other black-pigmented Porphyromonas and Bacteroides spp. The reactivity was also evidenced by immunoslot blot analysis (Fig. 2). These results show that BEEll antibody specifically recognizes P. endodontalis.

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HANAZAWA ET AL.

INFECT. IMMUN. Control

BEEIl - IP. ondodcontalis ATCC 35406

-

P.endocdor7talis HG 400

P.xingivaris

381

P.asaccharo/yticus ATCC; 25260

EO0.6

BHintermedius ATCC

C

25611

B.intermedius ATCC 25261

60.4 0~~~

B.melan'irnogonicus

ATCC 25845

0.2

D.Ioescheii ATCC 15930 0~~~~~

100

10

B.corporis ATCC 33547 1

0.1

Protein conc. (pg/ml) FIG. 1. Immunoreactivity of monoclonal antibody BEEll against black-pigmented Porphyromonas and Bacteroides spp. Immunoreactivity was determined by ELISA with sonicated bacterial extracts (protein concentration, 0.1 to 100 ,ug/ml) coated on NUNK microimmunoassay plates. The culture supernatant from BEEll antibody-producing cells was used in this experiment. Symbols: 0, P. endodontalis HG 400; 0, P. endodontalis ATCC 35406; B. *, P. gingivalis 381; A, P. asaccharolytica ATCC 25260; intermedius ATCC 25611; A, B. melaninogenicus ATCC 25845. O.D., Optical density. E,

BEEll antibody recognizes a serotype antigen of P. endodontalis. Next, we examined the reactivity of BEEll antibody toward several strains of P. endodontalis. As shown by immunoslot blot analysis (Fig. 3), the antibody reacted with strains ATCC 35406, HG 400, and HG 421 but not with HG 422 or HG 948. Therefore, this pattern suggests the possibility that BEEll antibody recognizes the 01K, antigen of the three serotypes of this bacterium designated by van Winkelhoff et al. (20). Reactivity of BEEll antibody against P. endodontalis cell components. Also using immunoslot blot analysis, we looked for the cellular location of the reactive antigen in P. endodontalis ATCC 35406. As. shown in Fig. 4, BEEll antibody reacted with the total cell envelope and capsular materials of the cells but not with the LPS component. Partial purification of a serotype antigen of P. endodontalis by BEEll antibody and characteristics of the antigen. We tried to partially purify the reactive serotype antigen of P. endodontalis. First, P. endodontalis ATCC 35406 sonicated extracts were fractionated on a Superose 12 prep-packed column. The fractions obtained were examined by ELISA for immunoreactivity with BEEll antibody. Figure Sa shows that the peak activity was detected in fractions corresponding to a molecular mass of 110 kilodaltons (kDa). The peak fraction was then subjected to SDS-PAGE and Western immunoblot analyses. By these procedures, the serotype antigen was detected as a single band, indicating an apparent molecular mass of approximately 52 kDa (Fig. Sb). These results suggest the possibility that the serotype antigen is a dimer consisting of two 52-kDa monomers. Next, the peak fraction from the Superose 12 prep chro-

FIG. 2. Immunoslot blot assay for reactivity of monoclonal antibody BEEll against black-pigmented Porphyromonas and Bacteroides spp. Sonicated bacterial extracts (protein concentration, 2 ,ug per well) were blotted onto nitrocellulose membranes in a Hybri-slot apparatus. The culture supernatants from SP2/0 Ag 14 myeloma (control) and BEEll antibody-producing cells were used in this experiment.

matography was pooled, concentrated, and then dialyzed against 20 mM Tris hydrochloride (pH 8.5). The dialyzed sample was then applied to a HPLC apparatus equipped with an IEC-DEAE Biofine column and eluted with a linear gradient of NaCl from 0 to 500 mM. The fractions were tested for reactivity toward BEEll antibody. Figure 6 shows that the peak fraction for the reactivity was eluted with 250 mM NaCl. This partially purified antigen was used for determination of sensitivity of the antigen to heat and enzymes. Effect of enzymes and heat treatment on a partially purified serotype antigen of P. endodontalis. The partially purified antigen was highly sensitive to trypsin and V8 protease. Control

BEEll

ATCC35406 HG400 HG421

HG422 HG948 FIG. 3. Immunoslot blot assay for reactivity of monoclonal antibody BEEll against several strains of P. endodontalis. Experimental conditions were as described in the legend to Fig. 2.

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MONOCLONAL ANTIBODY AGAINST P. ENDODONTALIS ANTIGEN

VOL. 58, 1990

Control

BEEII

B0.7 E 0.6 o 0.5 a 0.4 -

40M

sonicated extracts

total membrane

cmJ

0.40

0° 00.2-~~~~~~~~ 2L LPS capsular material

LL FIG. 4. Immunoslot blot assay for reactivity of monoclonal antibody BEEll against various cell components of P. endodontalis. The preparation of selected cell components of P. endodontalis ATCC 35406 is described in Materials and Methods. Test samples and their concentrations were as follows: sonicated extracts, 2 ,ug of protein per well; total membrane, 2 ,ug of protein per well; LPS, 2 ,ug per well; and capsular material, 0.2 ILg of hexose per well. Experimental conditions were as described in the legend to Fig. 2.

However, it showed resistance to digestion by neuraminidase. On the other hand, the antigen was quite sensitive to heating to 80°C. When the antigen was treated for 10 min at 60°C or 70°C, the reactivity was higher than that of the control (Table 2). We cannot now explain why the antigen expressed such higher antigenicity after exposure to these temperatures.

DISCUSSION Van Winkelhoff et al. (20) demonstrated the presence of three serotypes of P. endodontalis, which they designated as O1Kj, O1K2, and O1K-. However, the characteristics of these serotype antigens have not been defined. In the present study, we established a hybridoma cell line producing a monoclonal antibody (BEE11) that reacted with the O1Kj serotype antigen of P. endodontalis and then examined certain properties of the partially purified serotype antigen. ELISA and immunoslot blot analyses showed that this antibody reacted with various strains (ATCC 35406, HG 400, and HG 421) of P. endodontalis. BEEll monoclonal antibody proved to be highly specific for P. endodontalis, as it dp.>

15 20 Fraction number

FIG. 6. HPLC-DEAE chromatogram of the peak fraction obtained by Superose 12 prep chromatography. The peak fraction (0.5 mg of protein), with a molecular mass of 110 kDa, obtained by the gel filtration shown in Fig. 5 was applied to an HPLC apparatus equipped with an IEC-DEAE Biofine column. The immunoreactivity of each fraction toward BEEll antibody was determined by ELISA. O.D., Optical density.

did not react with any of the other black-pigmented Porphyromonas and Bacteroides species tested. It was recently suggested that LPS may be the common antigen for P. endodontalis (20). BEEll antibody did not react with LPS (Fig. 4). Therefore, these observations indicate that this monoclonal antibody recognizes the O1Kj serotype antigen of P. endodontalis but not the common antigen, LPS. Van Winkelhoff et al. (20) have suggested that two serotype antigens (01K1 and O1K2) of P. endodontalis may exist in the capsular layer. In the immunoslot blot analysis, BEEll antibody reacted with capsular materials from P. endodontalis ATCC 35406. This result supports their suggestion that the O0K1 serotype antigen may exist in the capsular layer of the organism. When the sonicated extracts were fractionated by Superose 12 prep gel chromatography, the serotype antigen was detected in fractions having a molecular mass of approximately 110 kDa. However, in Western blot analysis after SDS-PAGE, we observed that BEEll antibody reacted with a molecule having a molecular mass of approximately 52 kDa. These observations suggest the possibility that the serotype antigen is a dimer comprising two 52-kDa monomers. Although we do not yet know the structure of the antigen, SH bonds may not be involved in formation of the dimer because the SDS-PAGE of the

(b)

E 1.0 .(a) u"

~~5 10

TABLE 2. Effect of enzymes and heat on reactivity of BEEll antibody with partially purified antigen of P. endodontalis ATCC 35406

0.8 E

0. 6

74.4k& _ l, N .

04

0.8

Treatmenta _

*452kd

49.kd-

.~~~~~~~~0.4 37.2kdl-

W 0.2

-J~~~~~~~~~~~~~-.

20

40

60

80 100 120

Fraction number FIG. 5. Gel filtration of sonicated extracts of P. endodontalis ATCC 35406 by Superose 12 prep chromatography (a) and Western blotting assay (b). A sonicated extract (20 mg of protein) was applied to a Superose 12 prep-packed column. The elution conditions are described in Materials and Methods. The peak fraction (0.5 mg of protein), corresponding to a molecular mass of 110 kDa (110K), was used for the Western blotting assay as described in Materials and Methods. O.D., Optical density; kd, kilodaltons. Vo, Void volume.

Control .....................................

Immunoreactivity of control level) (% 100

Enzymes

Trypsin ..................................... V8 protease ....................

................. Neuraminidase .....................................

0 0 98

Heat

600C .......................................... 700C ..................................... 800C .....................................

150 146 0

a Partially purified antigen (50 p.g/ml) was treated separately with each enzyme (200 p.g/ml) for 10 min at 37°C or at various temperatures for 10 min. After treatment, immunoreactivity toward BEEll antibody was determined by ELISA.

2546

HANAZAWA ET AL.

antigen done in the absence of 2-mercaptoethanol also showed the 52-kDa protein. Further, the serotype antigen was partially purified by HPLC with an IEC-DEAE Biofinepacked column. The partially purified antigen was highly sensitive to trypsin and to V8 protease and quite sensitive to heating to 80°C. However, the antigen was not sensitive to neuraminidase. These findings suggest that protein contributes to the structure of the antigenic determinant. It has not yet been determined whether pathogenesis of P. endodontalis correlates with a given serotype of the bacterial cell. Monoclonal antibodies against each serotype antigen of the organism may provide excellent tools for an understanding of the roles played by the various serotypes in pathogenesis and for the quantitative monitoring of each serotype of P. endodontalis in odontogenic abscesses. Therefore, we are currently striving to establish monoclonal antibodies that recognize specifically the serotypes 01K2 and 01K- of P. endodontalis.

INFECT. IMMUN.

9.

10. 11.

12. 13.

14.

ACKNOWLEDGMENTS We thank T. J. M. van Steenbergen and J. de Graaff for providing

P. endodontalis. 15. 1. 2.

3. 4.

5.

6. 7. 8.

LITERATURE CITED Bradford, M. N. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein dye binding. Anal. Biochem. 72:248-254. Engvall, E., and P. Permann. 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. Haapasalo, M., H. Ranta, K. Ranta, and H. Shah. 1986. Blackpigmented Bacteroides spp. in human apical periodontitis. Infect. Immun. 53:149-153. Hanazawa, S., K. Nakada, Y. Ohmori, T. Miyoshi, S. Amano, and S. Kitano. 1985. Functional role of interleukin 1 in periodontal disease: induction of interleukin 1 production by Bacteroides gingivalis lipopolysaccharide in peritoneal macrophages from C3H/HeN and C3H/HeJ mice. Infect. Immun. 50:262-270. Hanazawa, S., K. Saitoh, Y. Ohmori, H. Nishihara, S. Fujiwara, and S. Kitano. 1984. Production of monoclonal antibodies that recognize specific and cross-reactive antigens of Bacteroides gingivalis. Infect. Immun. 46:285-287. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Martha, J. G., R. E. Corstvet, and R. J. Panciera. 1982. Extraction of capsular material from Pasteurella haemolytica. Am. J. Vet. Res. 43:2070-2073. Miyoshi, T., S. Hanazawa, K. Hirose, K. Saitoh, S. Amano, Y. Ohmori, and S. Kitano. 1986. Humoral antibody response

16. 17.

18.

19.

against Bacteroides gingivalis-specific antigen recognized by monoclonal antibody in adult periodontal patients. Infect. Immun. 53:366-371. Morris, D. L. 1948. Quantitative determination of carbohydrates with Dreywood's anthrone reagent. Science 107:254-255. Oguntebi, B., A. M. Slee, J. M. Tanzer, and K. Langeland. 1982. Predominant microflora associated with human periapical abscesses. J. Clin. Microbiol. 15:964-966. Oi, V. T., and L. A. Herzenberg. 1980. Immunoglobulin-producing hybrid cell lines, p. 351-357. In B. B. Mishell and S. M. Shiigi (ed.), Selected methods in cellular immunology. W. H. Freeman and Co., San Francisco. Pantera, E. A., J. J. Zambon, and M. Shin-Levine. 1988. Indirect immunofluorescence for the detection of Bacteroides species in human dental pulp. J. Endod. 14:218-223. Shah, H. N., and M. D. Collins. 1988. Proposal for reclassification of Bacteroides asaccharolyticus, Bacteroides gingivalis, and Bacteroides endodontalis in a new genus, Porphyromonas. Int. J. Syst. Bacteriol. 38:128-131. Slots, J. 1982. Importance of black-pigmented Bacteroides in human periodontal disease, p. 27-45. In R. J. Genco and S. E. Mergenhagen (ed.), Host-parasite interactions in periodontal diseases. American Society for Microbiology, Washington, D.C. Socransky, S. S. 1977. Microbiology of periodontal disease: present status and future considerations. J. Periodontol. 48: 497-504. Sundqvist, G., E. Johansson, and U. Sjogren. 1989. Prevalence of black-pigmented Bacteroides species in root canal infections. J. Endod. 15:13-19. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. van Steenbergen, T. J. M., A. J. van Winkelhoff, D. Mayrand, D. Grenier, and J. de Graaff. 1984. Bacteroides endodontalis sp. nov., an asaccharolytic black-pigmented Bacteroides species from infected dental root canals. Int. J. Syst. Bacteriol. 34: 118-120. van Winkelhoff, A. J., A. W. Carlee, and J. de Graaff. 1985. Bacteroides endodontalis and other black-pigmented Bacteroides species in odontogenic abscesses. Infect. Immun. 49:

494-497. 20. van Winkelhoff, A. J., N. Kippuw, and J. de Graaff. 1986. Serological characterization of black-pigmented Bacteroides endodontalis. Infect. Immun. 51:972-974. 21. van Winkelhoff, A. J., T. J. M. van Steenbergen, N. Kippuw, and J. de Graaff. 1985. Further characterization of Bacteroides

endodontalis, an asaccharolytic black-pigmented Bacteroides species from the oral S cavity. J. Clin. Microbiol. 22:75-79. 22. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharide extraction with phenol water and further applications of the procedures. Methods Carbohydr. Chem. 5:83-91.