An antigen recognized by monoclonal antibody specific for Bacteroides gingivalis was purified in the presence of 0.5% (wt/vol) j8-octyl-glucoside by ...
Vol. 53, No. 2
INFECTION AND IMMUNITY, Aug. 1986, p. 366-371
0019-9567/86/080366-06$02.00/0 Copyright © 1986, American Society for Microbiology
Humoral Antibody Response against Bacteroides gingivalis-Specific Antigen Recognized by Monoclonal Antibody in Adult Periodontal Patients TAKEHITO MIYOSHI,* SHIGEMASA HANAZAWA, KIMIHARU HIROSE, KIEKO SAITOH, SHIGERU AMANO, YOSHIHIRO OHMORI, AND SHIGEO KITANO Department of Oral Microbiology, Josai Dental University, Keyakidai, Sakado, Saitama 350-02, Japan Received 19 August 1985/Accepted 29 April 1986
An antigen recognized by monoclonal antibody specific for Bacteroides gingivalis was purified in the presence of 0.5% (wt/vol) j8-octyl-glucoside by immunoadsorbent column chromatography. The purified antigen was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining, and the pattern of SDS-PAGE agreed with that of immunoblotting. The molecule exhibited an apparent molecular weight of about 62,000 by SDS-PAGE. The antigen was sensitive to trypsin, Staphylococcus aureus V8 protease, DNase I and II, and heating, but insensitive to RNase and neuraminidase. By the enzyme-linked immunosorbent assay method, the purified antigen was not cross-reactive with rat polyclonal antibodies to each of several black-pigmented Baceroides species, Fusobacterium nucleatum, Eikenella corrodens, and ActinobaciUus actinomycetemcomitans. These results indicate that the purified antigen is specific for B. gingivalis. Humoral antibody titers in adult periodontal patients against the specific antigen were measured by the enzyme-linked immunosorbent assay. Serum immunogloblin G antibody titers against the specific antigen in adult periodontal patients correlated significantly with the severity of periodontal disease. However, such significant correlation was not observed with serum immunogloblin M antibody titers.
between serum IgG antibody titers against B. gingivalisspecific antigen in adult periodontal patients and the severity of the disease.
Bacteroides gingivalis is found in the humoral oral environment and has been isolated in relatively high proportions from inflamed gingival pockets of patients with adult periodontal disease (14, 15). Furthermore, extracellular protease and membrane-bound trypsin-like protease have been detected on B. gingivalis by several investigators (1, 20). Also, proteolytic activities against human protease inhibitors were detected in culture medium of this organism (3). All of these studies (1, 3, 11, 14, 15, 18) suggest that B. gingivalis may be associated with the pathogenesis of adult periodontal disease. Several investigations have indicated that immunogloblin G (IgG) serum antibody titers against B. gingivalis are increased significantly in adult periodontal patients (11, 16-18). However, the correlation between serum IgG or IgM antibody titers against B. gingivalisspecific antigens in periodontal patients and the severity of the disease has not been made definitely (4, 9). Therefore, it is very important for determination of the etiology of adult periodontal disease to verify such a correlation. For clinical studies of adult periodontitis, it is important to rapidly detect and identify B. gingivalis in the oral cavity. Specific and highly sensitive markers of B. gingivalis would be of great use to allow us to establish simple methods for clinical investigation. B. gingivalis is considered to be antigenically distinct from other Bacteroides species (12). However, it has not been studied in detail. Hanazawa et al. (6) have reported the production of monoclonal antibodies specific for B. gingivalis by the fusion of SP2/0 Agl4 myeloma cells to spleen cells from BALB/c mice immunized with B. gingivalis 381. We describe here the purification and some properties of the antigen recognized by one monoclonal antibody (BGF7) specific for B. gingivalis (6) and discuss its specificity. Furthermore, we observed a significant positive correlation
MATERIALS AND METHODS Bacterial strain. B. gingivalis 381 was used in the present study. Also, B. gingivalis ATCC 33277, Bacteroides asaccharolyticus ATCC 25260, Bacteroides intermedius ATCC 25261 and ATCC 25611, Bacteroides melaninogenicus ATCC 25845 and 1-13K-2, Bacteroides corporis ATCC
33547, Fusobacterium nucleatum (oral isolate), Eikenella corrodens ATCC 23834, and Actinobacillus actinomycetemcomitans ATCC 29522 were used for preparation of polyclonal antibodies. EX-1 diffusate medium (10) was used for the cultivation of Bacteroides strains and A. actinomycetemcomitans. E. corrodens was grown in medium which contained the following: 2% (wt/vol) Trypticase soy (BBL Microbiology Systems, Cockeysville, Md.), 0.02% (wt/vol) KNO3, 5% (wt/vol) yeast extract, and 5 p,g of hemin per ml. Bacteroides strains, F. nucleatum, and E. corrodens were cultivated for 24 to 48 h at 37°C under anaerobic conditions, and A. actinomycetemcomitans was grown for 4 days at 37°C in a 20% (wt/vol) CO2 incubator. These strains were harvested, washed with phosphate-buffered saline (PBS), and lyophilized. Preparation and purification of BGF7 monoclonal antibody from ascitic fluid. BGF7 monoclonal antibody-producing cells (1 x 107 cells per mouse) were injected intraperitoneally into BALB/c mice which had been treated with 1.0 ml of pristane (2,6,10,14-tetramethylpentadecane; Aldrich Chemical Co., Inc., Milwaukee, Wis.) 7 days before. The ascitic fluid in each mouse was collected and pooled. The cells and debris were removed from the ascitic fluid by centrifugation at 1,000 x g for 10 min, and the supernatant was frozen and stored at -20°C. After thawing, additional fibrin and partic-
* Corresponding author. 366
VOL. 53, 1986
ulate material were removed by centrifugation at 10,000 x g for 20 min. The ascitic fluid thus prepared was used for the enzyme-linked immunosorbent assay (ELISA) and the immunoblotting assay. The ascitic fluid (10 ml) was diluted with 4 volumes of 20 mM Tris hydrochloride (pH 7.5), and an equal volume of saturated ammonium sulfate solution at room temperature was added slowly with vigorous stirring. The suspension was kept on ice for 60 min and then centrifuged at 10,000 x g for 10 min. The pellet was dissolved in 20 mM Tris hydrochloride (pH 7.5), and the ammonium sulfate precipitation was repeated two more times under the same conditions. The final pellet was suspended in 20 mM Tris hydrochloride (pH 7.5) and dialyzed for 24 h against the same buffer. The dialyzed solution was centrifuged at 15,000 x g for 10 min. The supernatant was adsorbed to a DEAE Affi-gel Blue (Bio-Rad Laboratories, Rockville Center, N.Y.) column (1.5 by 12 cm) equilibrated with 20 mM Tris hydrochloride (pH 7.5) (2). The column was washed with the same buffer, and the proteins were eluted with a linear NaCl gradient (0 to 200 mM) in the same buffer. The IgG fractions were pooled and used as purified monoclonal antibody. Preparation of immunoadsorbent. The purified monoclonal antibody was lyophilized, dissolved in coupling buffer (0.1 M NaHCO3 containing 0.5 M NaCI), and dialyzed for 24 h against the same buffer. The dialyzed IgG solution was diluted to a protein concentration of 2.0 mg/ml with the coupling buffer and then mixed with CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) (1 g of gel [dry weight] per mg of protein) washed previously with 10-3 M HCl. The mixture was rotated end-over-end for 2 h at room temperature. After the reaction, the gel was washed with the coupling buffer, suspended in 1 M ethanolamine hydrochloride (pH 8.0), and reacted with this reagent for 1 h at room temperature. Thereafter, the washing of the gel with 0.1 M acetate buffer (pH 4.0) containing 0.5 M NaCl and with 0.1 M Tris hydrochloride (pH 8.0) containing 0.5 M NaCl was repeated three times. The gel was finally rinsed with 20 mM Tris hydrochloride (pH 7.5) and stored in the same buffer containing 0.02% (wt/vol) NaN3 at 4°C. SDS-PAGE. The antigen materials were boiled for 3 min in 1.25 mM Tris glycerol (pH 8.3) containing 1% (wt/vol) sodium dodecyl sulfate (SDS), 5% (vol/vol) 2-mercaptoethanol, and 25% (vol/vol) glycerol. The boiled materials were separated by SDS-polyacrylamide gel electrophoresis (PAGE) by using the method of Laemmli (8). The materials were stacked in an 8% (wt/vol) polyacrylamide gel and separated on a 10% (wt/vol) gel. After SDS-PAGE, proteins on the gel were visualized by silver staining (Ag-Stain Daiichi; Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan). Immunoblotting. The antigen materials were applied to SDS-PAGE. After SDS-PAGE, the separated samples were transferred to nitrocellulose paper by the method described by Towbin et al. (19). After transfer, the antibody binding was visualized by using horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) and HRP Color Development Reagent (Bio-Rad). Enzymes and heat treatments. A 10-,ul amount of each enzyme solution was added to separate tubes containing 20 ,ul of the antigen materials diluted to 90,ul with 50 mM Tris hydrochloride (pH 6.8). Trypsin (Type I; Sigma Chemical Co., St. Louis, Mo.), Staphylococcus aureus V8 protease (Miles Laboratories, Inc., Elkhart, Ind.), RNase (P-L Biochemicals Inc., Milwaukee, Wis.), DNase I (Type IV; Sigma), and DNase II (Type IV; Sigma) were used at a final concentration of 200 pLg/ml; and neuraminidase (Type IV;
B. GINGIVALIS-SPECIFIC ANTIGEN
367
Sigma) was used at a final concentration of 100 ,ug/ml. After incubation for 10 min at 37°C, the treated antigen materials were diluted with cold coating buffer (50 mM sodium carbonate [pH 9.6] containing 0.02% [wt/vol] NaN3) for the ELISA. For the measurement of heat stability, the antigen materials were incubated for 10 min at 60, 70, or 80°C and thereafter rapidly cooled on ice. ELISA. The ELISA was performed by a modification of the method of Engvall and Perlmann (5). For the detection of the antigen at each purification step, 100 1.I of test sample was coated onto the well walls of Falcon plastic microtiter plates (Becton Dickinson and Co., Oxnard, Calif.) by an overnight incubation at 4°C. The wells were washed with PBS containing 0.02% (vol/vol) Tween 20 (PBS-Tween) and then loaded with 100 RI of 5% (wt/vol) bovine serum albumin in PBS containing 0.02% (wt/vol) NaN3. After incubation for 2 h at room temperature, the wells were washed with PBS-Tween. Portions (100 ,u) of the monoclonal antibody BGF7 appropriately diluted with PBS-Tween were added and incubated for 1 h at room temperature. The ascitic fluid from BALB/c mice injected with SP2/0 myeloma cells was diluted with PBS-Tween and used as a control. After the wells were washed with PBS-Tween, 100 ,u of the alkaline phosphatase-conjugated goat anti-mouse IgG (Cooper Biomedical, Inc., West Chester, Pa.) diluted with PBS-Tween was added to each well. After another incubation for 1 h at room temperature, the wells were washed. Then 100 ,u of p-nitrophenylphosphate (1 mg/ml; Sigma) in diethanolaminehydrochloride (pH 9.8) containing 0.02% (wt/vol) NaN3 and 0.5 mM MgCl2 was added, and the optical density at 405 nm was spectrophotometrically measured. For detection of humoral antibodies, the purified antigen was appropriately diluted with coating buffer and coated onto well walls of plastic microtiter plates during an overnight incubation at 4°C. After the treatment of wells with bovine serum albumin, 100 RI of serum from each subject diluted with PBS was added as the first antibody, and the plates were incubated for 1 h at room temperature. PBS was used as a negative control. After the wells were washed with PBS-Tween, 100 RI of alkaline phosphatase-conjugated goat anti-human IgG (E.Y. Laboratories, Inc., San Mateo, Calif.) or anti-human IgM (Tago Inc., Burlingame, Calif.) diluted with PBS-Tween was added to each well as the second antibody. After another incubation for 1 h at room temperature, the wells were washed. p-Nitrophenylphosphate was used for visualizing the antibody binding. Other procedures were carried out as described above. To assay the reactivities of the purified antigen with the B. gingivalis-specific monoclonal antibodies and with polyclonal antibodies directed against other oral bacteria, the ELISA was also used. The purified antigen or the cell homogenate of B. gingivalis was coated onto the well walls of a plastic microtiter plate during an overnight incubation at 4°C. Culture supernatants of two monoclonal antibodyproducing cell lines (BGF7 and BGD2) and rat polyclonal antibodies against each bacterial strain were used separately as the first antibody. After the treatment of wells with bovine serum albumin, 100 plI of a given culture supernatant or polyclonal antibodies diluted with PBS was loaded into the wells. Horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) and anti-rat IgG (Cooper Biomedical) and HRP Color Development Reagent (Bio-Rad) were used for visualizing the antibody binding. The optical density at 590 nm was spectrophotometrically measured. Other procedures were performed as described above. Preparation of polyclonal antibodies. Each lyophilized bac-
368
MIYOSHI ET AL.
INFECT. IM MUN.
terial strain (dry weight, 5 mg) was suspended in 2 ml of PBS and then mixed with 2 ml of Freund complete adjuvant (Difco Laboratories, Detroit, Mich.). These mixed suspensions were sonicated and used as immunogen. The bacterial suspensions were injected intraperitoneally into rats (6 weeks old). After 2 weeks, the same dosage of each bacterial strain, but this time in PBS, was again intraperitoneally injected into the rats. A trial bleeding was taken at 7 days after the second injection. Serum was tested for specific antibodies by immunodiffusion. Over 2 weeks after the second injection, a third injection in PBS was given intraperitoneally, and the rats were finally exsanguinated via cardiac puncture 4 days thereafter. Purification of specific antigen from cell envelope. For the purification of the antigen, B. gingivalis 381 was grown anaerobically for 24 h at 37°C in EX-1 diffusate medium (10). The harvested cells which were washed with 20 mM Tris hydrochloride (pH 7.5) and suspended in the same buffer were used as the starting material. The antigen not only was distributed in the cell envelope, but was also detected in the culture medium as well as in the cytoplasm by the ELISA (data not shown). For the studies described in this report, we used 0.5% (wt/vol) f3-octyl-glucoside (Sigma) and solubilized the antigen from the cell envelope fraction. All purification
170,000 _
97,40 0_
5 5,400_
36,500 _
20,100O
A
B
FIG. 1. SDS-PAGE and immunoblotting of purified antigen. Lane A, silver staining of purified antigen after SDS-PAGE. Lane B, monoclonal antibody BGF7 detection of purified antigen. The arrows pointing to the right show the positions of proteins used as apparent molecular weight markers, whereas that pointing left (lane B) indicates the position of the immunoreactive band.
TABLE 1. Stability of purified antigen to various enzymes and heat treatment Treatment
Residual reactivity with
monoclonal antibody (BGF7) (%)
Control ..................................... 100 Enzymes
Trypsin ................................... 0.0 V8 protease ................................ DNase I ................................... DNase II .................................. RNase .................................... Neuraminidase .............................
Heating (°C) 60 ...................................... 70 ..................................... 80 .....................................
71.1 13.0 10.6 92.7 95.8 98.9 95.8 31.2
procedures described below were carried out at 4°C or below. The cell suspension was sonicated, and unbroken cells and debris were removed by centrifugation at 10,000 x g for 30 min. The supernatant was further centrifuged at 80,000 x g for 90 min. The pellet, containing the cell envelopes, was suspended in 20 mM Tris hydrochloride (pH 7.5) containing 0.5% (wt/vol) 3-octyl-glucoside (buffer A) and solubilized for 30 min with vigorous stirring. The solubilized material was centrifuged at 80,000 x g for 90 min, and the pellet was solubilized again under the same conditions with 0.5% (wt/vol) p-octyl-glucoside. The above solubilized materials were combined and concentrated by polyethylene glycol. The concentrated antigen solution was loaded onto an immunoadsorbent column (1.5 by 10 cm) made with purified monoclonal antibody BGF7 and equilibrated with buffer A containing 0.5 M NaCl (buffer B). After being washed with 15 column volumes of buffer B, the column was rinsed with 7 column volumes of 0.5% (wt/vol) 3-octyl-glucoside containing 0.2 M NaCl and then eluted with 0.2 N acetic acid (pH 2.5) containing 0.5% (wt/vol) ,B-octyl-glucoside and 0.2 M NaCl (buffer C). The fractions eluted with buffer C were immediately neutralized with 2 M Tris hydrochloride (pH 8.0). At the end of the elution, the column was washed with 80 mM Tris hydrochloride (pH 7.5). The pooled antigen was concentrated with polyethylene glycol and a Minicon Concentrator (Amicon Corp., Lexington, Mass.) and dialyzed against the coating buffer used for ELISA. The dialyzed antigen solution served as the purified antigen in the following experiments. Sera from adult periodontal patients. Periodontal subjects (age range, 20 to 64 years) at Josai Dental University Hospital were characterized by the periodontal index (PI) described by Russell (13). Control subjects (PI-' 0.1) were healthy individuals who had not received dental treatment for at least 7 months before this study. All sera were stored at -70°C until used. RESULTS Purity and immunoblotting of purified antigen. The antigen purified from B. gingivalis cell envelopes by the BGF7 monoclonal antibody immunoadsorbent column was subjected to SDS-PAGE and Western immunoblot analyses. The purified antigen was homogeneous as judged by the silver staining after SDS-PAGE (Fig. 1, lane A) and was detected as a single band with the same mobility (Fig. 1, lane
VOL. 53, 1986
B. GINGIVALIS-SPECIFIC ANTIGEN
TABLE 2. Reactivities of purified antigen with monoclonal antibodies specific for B. gingivalis and with polyclonal antibodies against other Bacteroides species and oral bacteria Antibody
Monoclonal antibodies BGF7 BGD2
Polyclonal antibodies against B. gingivalis 381 B. gingivalis ATCC 33277 B. intermedius ATCC 25611 B. intermedius ATCC 25261 B. melaninogenicus ATCC 25845 B. melaninogenicus 1-13K-2 B. asaccharolyticus ATCC 25260 B. corporis ATCC 33547 E. corrodens ATCC 23834 F. nucleatum (oral isolate) A. actinomycetemcomitans ATCC 29522
Reactivity with: Purified Cell antigen homogenate
+ +
+ +
+ + -
+ + -
-
-
-
-
-
-
-
-
-
-
-
-
369
correlation in the patients having a PI of 0.0 to 4.0 was more significant than that in the patients with a PI of 0.0 to 8.0. However, no such correlation was observed when serum IgM antibody titers against the specific antigen were examined (Fig. 3). Relation between serum antibody titers and age of subjects. Several investigators (4, 7) have reported evidence that the level of serum IgG antibody against B. gingivalis is positively correlated with age. When the IgG antibody titers of the tested periodontal subjects were plotted against age, however, no correlation was found (Fig. 4). Thus, the increase in serum IgG antibody titer against B. gingivalisspecific antigen does not appear to be related to age.
DISCUSSION Recently, Hanazawa et al. (6) reported the development of monoclonal antibodies specific for Bacteroides gingivalis. In the present study, we purified (to apparent homogeneity) the
1.0 0.8
B). However, a major band and a few minor ones reactive with monoclonal antibody BGF7 were observed in the immunoblotting after SDS-PAGE of the cell homogenate (data not shown). These minor bands were removed in the process of the purification. Why the monoclonal antibody reacted with these minor bands is not clear. The purified antigen exhibited an apparent molecular weight of approximately 62,000 as judged from its mobility on the SDS-PAGE. Effect of enzymes and heat treatment on purified antigen. The purified antigen was highly sensitive to trypsin, moderately so to V8 protease, and quite sensitive to heating at 80°C (Table 1). Also, DNase I and II inactivated it. DNase I was substantially free of RNase and protease, and DNase II was free of RNase (Sigma; information from manufacturer). On the other hand, the antigen was not sensitive to RNase or neuraminidase, but the participation of saccharides in the antigenic site is not ruled out because of the substrate specificity of neuraminidase. These results suggest the possibility that polypeptides and deoxyribonucleotides contribute to the antigenic determinant. Specificity of purified antigen. The monoclonal antibody (BGF7) used for the purification of the antigen was specific for B. gingivalis (6). However, the other sites on the antigen molecule, except the antigenic determinant of the monoclonal antibody (BGF7), may be cross-reactive with other bacteria. Thus, the reactivity of the purified antigen with polyclonal antibodies against other oral bacteria was also examined. The purified antigen was reactive with the monoclonal antibodies specific for B. gingivalis and polyclonal antibodies to it (Table 2). However, the purified antigen did not react with rat polyclonal antibodies against other Bacteroides species and other oral bacteria. Serum IgG and IgM antibody titers against purified antigen. A total of 46 subjects were divided into healthy subjects (PI ' 0.1), PI-1 group (0.1 < PI-' 2.0), PI-2 group (2.0 < PI ' 4.0), and PI-3 group (4.0 < PI -' 8.0). The serum IgG antibody titers of PI-2 and PI-3 groups against the specific antigen were significantly higher than those of healthy subjects (Fig. 2). The correlation between serum IgG antibody titers against the specific antigen and PI of all the patients was positive (r = 0.35; P < 0.05; Fig. 2, inset). When just the 32 subjects with a PI of 0.0 to 4.0 were analyzed, the correlation coefficient (r) was 0.58 (P < 0.05). Thus, the
0
0.6
E
0.81
0
0
0.4 0
*
ci
.S.S e
*
0
S.*..
o 0.2
0
2 4 6 Periodontal Index
8
0
.
0.61* E
0
C
0
L()
0
0
,d 0
0.41*
0 0
.
0 0
0
I 0 0
0.2
0 0
0
0 0
0
Healthy Pl-1 Pl-2 Pl-3 Subjects FIG. 2. Relationship between serum IgG antibody titers against B. gingivalis-specific antigen and severity of adult periodontitis. 0 and Means and standard errors, respectively. The optical density of the wells loaded with PBS instead of sera was subtracted from each titer of subjects. Inset, Serum IgG antibody titers plotted against periodontal index of adult periodontal patients. O.D., Optical density. -,
370
MIYOSHI ET AL.
INFECT. IMMUN.
B. gingivalis antigen reactive with one of these monoclonal antibodies (designated BGF7). We do not clearly know at present where the specific antigen is localized in the cell. The antigen was detected in the sediment of sonicated extracts prepared by centrifugation at 80,000 x g. Then we purified the antigen from this sediment in the presence of ,B-octylglucoside. The antigen was sensitive to heating as well as to digestion by proteases and DNases (Table 1). Thus, polypeptides and deoxyribonucleotides appear to be involved in its antigenic determinant. It is known that the serum IgG antibody titers against B. gingivalis are significantly elevated in adult periodontal patients. Mansheim et al. (9) suggested previously that the serum IgG levels were not significantly elevated with the degree of periodontitis, but rather with the age of the subjects. However, crude antigens (e.g., cell homogenate or capsular materials) of B. gingivalis were used in these studies (9, 11, 18). Reed et al. (12) suggested that B. gingivalis should be considered to be antigenically distinct from oral and nonoral strains of B. asaccharolyticus and B.
1.0 A
0.8. A
0
0.6 I
E
ci 6
1.0
A
A
Al
In 0 A
A
A
.
A
0
0.4 .
0.0
A
A
A
0 0
8
AA
A
0 0
0.8
o
A
a
0
0.21
A
0
0 A
0
0
A 0
0.6 0
Ec 0 le.
6i 05
*T
.
0
0.4
0 0
2
;L7T
* f1?
I
£ 1
0
0.2I
*
o a *
Pl-2
Pl-3
a 0 0 0
Healthy Subjects
Pl-1
FIG. 3. Relationship between serum IgM antibody titers against B. gingivalis specific antigen and severity of adult periodontitis. O and Means and standard error*s, respectively. The optical density of the wells loaded with PBS inste.ad of sera was used for a reference as described in Materials and Meethods. O.D., Optical density. -,
20
30
40 Age
50
60
FIG. 4. Relationship between serum IgG antibody titers against B. gingivalis-specific antigen and age of subjects. IgG antibody titers of healthy subjects (0), subjects in PI-1 group (0), subjects in PI-2 group (A), and subjects in PI-3 group (A) are plotted against age.
O.D., Optical density.
melaninogenicus subspecies (recently referred to as B. asaccharolyticus, B. intermedius, B. melaninogenicus, and Bacteroides levii; see reference 7). However, they stated in their report that some strains of B. gingivalis weakly reacted with rabbit antisera to whole cells of nonoral B. asaccharolyticus (B. asaccharolyticus). Hanazawa et al. earlier reported a monoclonal antibody that recognized a cross-reactive antigen between B. gingivalis and B. intermedius (6). Thus, the antigenicity of B. gingivalis is not clear. These findings suggested that antibody against other bacteria, those with cross-reactive antigens, might be measured along with antibody against B. gingivalis when the serum titers of antibody against B. gingivalis in adult periodontal patients are measured by the ELISA method. It is important for the above reason that specific antigens of B. gingivalis be used for the measurements of the serum antibody titers in periodontal patients. The purified antigen described here seems to be suitable for the measurement of serum antibody titers against B. gingivalis since the mono-
VOL. 53, 1986
clonal antibody BGF7, with which it reacts, is specific for B. gingivalis and not cross-reactive with B. intermedius, B. melaninogenicus, and B. asaccharolyticus (6). Furthermore, we have shown by the ELISA method that the purified antigen was not cross-reactive with rat polyclonal antibodies directed against B. intermedius, B. asaccharolyticus, B. corporis, B. melaninogenicus, F. nucleatum, E. corrodens, and A. actinomycetemcomitans (Table 2). The increase in titer of serum IgG antibodies in the patients used in our present study reflects an increase in those specific for B. gingivalis since the antigen used in the test was specific for this organism. Since B. gingivalis may be associated with the pathogenesis of adult periodontal disease, this antigen should be a useful tool for determination of the etiology of adult periodontal disease. Further chemical and biochemical analyses of the antigen may justify such an application. On the other hand, significantly low antibody titers were observed in some patients With a PI of 4.0 to 8.0 (Fig. 2, inset). We do not know why serum antibodies in these subjects exhibited such low titers. However, this may reflect the presence of immune complexes in the sera of these patients, the degree of B. gingivalis infection, or the genetic variation among subjects in response to the antigen, etc. Since the ages of the healthy subjects examined were the ages 20 and 32 years, whereas of the patients were widespread, it is necessary to examine serum antibody levels in 30- to 50-year-old healthy subjects. We would like to study these problems in the future. ACKNOWLEDGMENTS We thank S. Tanaka and T. Masuda (Department of Oral Diagnosis, Josai Dental University) for supplying the sera from patients and for their kind suggestions. Also, we thank L. D. Frye for reviewing the manuscript. This work was supported by grants from the Scientific Research Funds (no. 60570876 and no. 60771535) of the Ministry of Education, Science and Culture of Japan. LITERATURE CITED 1. Abiko, Y., M. Hayakawa, S. Murai, and H. Takiguchi. 1985. Glycylprolyl dipeptidylaminopeptidase from Bacteroides gingivalis. J. Dent. Res. 64:106-111. 2. Bruck, C., D. Portetelle, C. Glineur, and A. Bollen. 1982. One-step purification of mouse monoclonal antibodies from ascitic fluid by DEAE Affi-gel blue chromatography. J. Immunol. Methods 53:313-319. 3. Carlsson, J., B. F. Herrmann, J. F. Hofling, and G. K. Sundqvist. 1984. Degradation of the human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis. Infect. Immun. 43:644-648. 4. Doty, S. L., D. E. Lopatin, S. A. Syed, and F. N. Smith. 1982. Humoral immune response to oral microorganisms in periodontitis. Infect. Immun. 37:499-505. 5. 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.
B. GINGIVALIS-SPECIFIC ANTIGEN
371
Immunol. 109:129-135. 6. 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. 7. Holdeman, L. V., R. W. Kelley, and W. E. C. Moore. 1984. Genus I. Bacteroides Castellani and Chalmer 1919, 959, p. 604-631. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 8. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 9. Mansheim, B. J., M. L. Stenstrom, S. B. Low, and W. B. Clark. 1980. Measurement of serum and salivary antibodies to the oral pathogen Bacteroides asaccharolyticus in human subjects. Arch. Oral Biol. 25:553-557. 10. Mashimo, P. A., and S. A. Ellison. 1972. Diffusate medium for cultivation of anaerobic bacteria. J. Dent. Health 22:38-45. 11. 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. 12. 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. 13. Russell, A. L. 1956. A system of classification and scoring for prevalence surveys of periodontal disease. J. Dent. Res. 35:350-359. 14. 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 disease. American Society for Microbiology, Washington, D.C. 15. Slots, J., and R. J. Genco. 1984. Black-pigmented Bacteroides species, Capnocytophaga species and Actinobacillus actinomycetemcomitans in human periodontal disease: virulence factors in colonization, survival and tissue destruction. J. Dent. Res. 63:412-421. 16. Taubman, M. A., J. L. Ebersole, and D. J. Smith. 1982. Association between systemic and local antibody and periodontal disease, p. 283-298. In R. J. Genco and S. E. Mergenhagen (ed.), Host-parasite interactions in periodontal disease. American Society for Microbiology, Washington, D.C. 17. Tew, J. G., D. R. Marshall, W. E. C. Moore, A. M. Best, K. G. Palcanis, and R. R. Ranney. 1985. Serum antibody reactive with predominant organisms in the subgingival flora of young adults with generalized severe periodontitis. Infect. Immun. 48: 303-311. 18. Tolo, K., and P. Brandtzaeg. 1982. Relation between periodontal disease activity and serum antibody titers to oral bacteria, p. 270-282. In R. J. Genco and S. E. Mergenhagen (ed.), Hostparasite interactions in periodontal disease. American Society for Microbiology, Washington, D.C. 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. 20. Yoshimura, F., M. Nishikata, T. Suzuki, C. I. Hoover, and E. Newbrun. 1984. Characterization of trypsin-like protease from the bacterium Bacteroides gingivalis isolated from human dental plaque. Arch. Oral Biol. 29:559-564.
