A novel approach for detecting an immunodominant antigen of Porphyromonas gingivalis in diagnosis of adult periodontitis

Abstract

In the course of long-term infection with Porphyromonas gingivalis in adult periodontitis, a specific antibody response to this organism is generated. We describe a potential novel approach for identifying an immunodominant antigen in human periodontitis patients. First, various monoclonal antibodies (MAbs) were established from mice immunized with crude antigen preparations of P. gingivalis FDC 381. The antigen specificities of these MAbs were compared with those of serum antibodies of 10 periodontitis patients in a competitive enzyme-linked immunosorbent assay. The binding of one MAb (termed PF18) was readily inhibited by sera from all patients but not by sera from healthy volunteers. The antigen recognized by PF18 existed on the cell surface, presumably in the capsule layer, shown by immunoelectron microscopic analysis. Purification of the antigenic substance, termed PF18-Ag, was performed by immunoaffinity chromatography with the MAb. Characterization of PF18-Ag suggested that the epitope was composed of carbohydrates but not peptides and that the substance was different from lipopolysaccharide. Measurement of levels of serum antibody to PF18-Ag better discriminated periodontitis patients from healthy individuals than measurement of antibodies to crude antigen preparations of P. gingivalis. Immunoglobulin G2 was the predominant isotype among the antibodies to PF18-Ag in the patients' sera. These results suggest that PF18-Ag, which is possibly a novel substance, is an important antigenic substance and is potentially useful for the clinical diagnosis of adult periodontitis. The approach that was used would also be relevant to detecting immunodominant antigens of other infectious microorganisms.

FIG. 1

Antigen specificities of MAbs in immunoblotting analysis. SDS-PAGE was performed with a cell-free sonicated extract of P. gingivalis FDC 381, and the resolved substances were transferred to nitrocellulose membranes. The antigen specificities of 19 MAbs which had shown positive reactions in ELISA were analyzed. One MAb that was positive for IgG production but not for anti-P. gingivalis activity (lane 20; MAb PS25) was also included as a negative control. The lane marked Control was not incubated with MAb but was incubated with the AP-conjugated second antibody. The membrane strips saturated with 1% bovine serum albumin were incubated with undiluted culture supernatants of hybridomas. MAbs which showed the same staining profiles were grouped into four types, A, B, C, and D, as indicated at the bottom. Other MAbs that showed unique patterns or no reactions were left ungrouped. The nomenclature used for the MAbs was from the bacterial preparations used to immunize mice: PF, MAb established from mice immunized with formalin-fixed cells; PS, sonic extracts; PA, autoclaved extracts. KD, kilodaltons.

FIG. 2

Inhibition of MAb reaction by antibodies from sera from patients with periodontitis. Sera from 10 patients diluted 1/10, 1/100 and 1/1,000 in PBS-T were added to microtiter plates which were precoated with sonicated P. gingivalis, and the plates were incubated for 30 min at 25°C. After removal of unbound antibodies, the plates were incubated with four different MAbs: PF18 (A), PS2 (B), PA20 (C), and PF24 (D). The MAbs were diluted to concentrations that gave half-maximum binding without inhibitors, which were determined in preliminary experiments. The binding of MAbs was determined by using a mouse IgG-Fc-specific antibody conjugated with AP (no cross-reactivity to human immunoglobulins). Binding of the MAbs was expressed as follows: (mean A₄₀₅ for triplicate test wells incubated with human serum prior to the incubation with MAbs/mean A₄₀₅ for triplicate control wells without human sera) × 100 (to give a percent).

FIG. 3

Marginal inhibition of MAb PF18 reaction by antibodies from sera from healthy individuals. A competitive inhibition assay (ELISA) was performed for MAb PF18 and sera from 10 healthy individuals as described in the legend to Fig. 2.

FIG. 4

Immunoaffinity purification of PF18-Ag and its profile by SDS-PAGE. (A) Sonic supernatants of P. gingivalis FDC 381 were subjected to a column of PF18-coupled Sepharose. After extensive washing with PBS and PBS containing 0.5 M NaCl, the substance that specifically bound to the MAb was obtained by acid elution. The concentration of protein in each fraction was monitored by measuring the A₂₈₀ (dotted line), and the activity as the ligand for MAb PF18 was determined by a sandwich ELISA as described in Materials and Methods (solid line). (B) SDS-PAGE profiles of various preparations of P. gingivalis FDC 381. Sonic extract (lane 1), affinity-purified PF18-Ag (lane 2), fimbriae (lane 3), and LPS (lane 4) were subjected to SDS-PAGE under reducing conditions, and the gel was developed by silver staining. Relative molecular masses obtained by using a molecular mass marker (Pharmacia) are indicated on the left.

FIG. 5

Effects of treatment with periodate or trypsin on antigenicities of PF18-Ag and fimbrillin. Reaction of MAb PF18 to modified PF18-Ag (A), reaction of a rabbit antifimbrillin antibody to modified fimbriae (B), and reaction of PF18 to unmodified fimbriae (C) were examined in immunoblot analyses. Lane 1, unmodified antigens; lane 2, antigens treated with periodate; lane 3, antigens treated with trypsin. The strips of the membrane were incubated with PF18 (5 μg/ml) followed by an incubation with AP-conjugated anti-mouse IgG-Fc (1/2,000; A and C) or with the rabbit antibody (diluted 1/500), followed by incubation with AP-conjugated anti-rabbit IgG (1/3,000; B).

FIG. 6

Localization of PF18-Ag by transmission electron microscopy. P. gingivalis FDC 381 cells were incubated with MAb PF18 and probed with gold-labeled protein A (diameter, 7 nm).

FIG. 7

Levels of antibody to affinity-purified PF18-Ag and to a crude antigen preparation of P. gingivalis FDC 381 in human sera. Serially diluted sera from 10 patients with adult periodontitis (P) and 10 healthy volunteers (H) were added to microtiter plates coated either with purified PF18-Ag (1 μg/ml) or with sonicated P. gingivalis FDC 381. The bound antibodies were probed with an AP-conjugated goat antibody with specificity for human IgG and IgM. The dose-response binding curves for individual serum samples were approximated by logistic curves, and the dilution factors at half-maximum binding on the logistic curves were compared with each other. Relative antibody titers for each serum sample were calculated by comparing the dilution factor with that for serum from one of the patients who showed the highest antibody activity both for PF18-Ag and for the crude antigen preparation, whose antibody titers for the two antigens were regarded as 100 ELISA units (EU). Each circle represents a result for one subject. Background values (broken horizontal lines), median values (thick horizontal lines), 25 and 75% fractiles (solid vertical lines with thin horizontal lines), and 10 and 90% fractiles (broken vertical lines with thin horizontal lines) are indicated.

FIG. 8

IgG subclass distribution of antibody to PF18-Ag in sera from patients. Serially diluted sera were added to microtiter plates precoated with PF18-Ag and were probed with MAb specific for each of four human IgG subclasses. The reactions were followed by incubation with AP-conjugated anti-mouse IgG-Fc (no cross-reaction to human immunoglobulins). Dose-response binding curves for each IgG subclass for individual patients were obtained by logistic approximation, as described in the legend to Fig. 7. The relative antibody activities of the different IgG subclasses in different individuals were calculated by comparing the dilution factors at half-maximum binding with that of IgG1 from the patient who showed the highest IgG antibody level (patient 1) (Fig. 7). The concentrations of the IgG subclass-specific MAb were adjusted to develop an equivalent absorbance when they were added to an equal amount of relevant IgG subclasses coating an ELISA plate to allow for an approximate comparison between levels of antibodies of the different IgG subclasses. The IgG1 activity of patient 1 was regarded as 100 ELISA units (EU). The numbers 1 to 10 at the bottom indicate the different patients.