Serum immunoglobulin G (IgG) and IgG subclass responses to the RgpA-Kgp proteinase-adhesin complex of Porphyromonas gingivalis in adult periodontitis

Abstract

Serum immunoglobulin G (IgG), IgM, and IgG subclass responses to the RgpA-Kgp proteinase-adhesin complex of Porphyromonas gingivalis were examined by enzyme-linked immunosorbent assay using adult periodontitis patients and age- and sex-matched controls. Twenty-five sera from subjects with adult periodontitis (diseased group) and 25 sera from healthy subjects (control group) were used for the study. Sera and subgingival plaque samples from 10 sites were collected from each patient at the time of clinical examination. The level of P. gingivalis in the plaque samples was determined using a DNA probe. Highly significant positive associations between the percentage of sites positive for P. gingivalis and measures of disease severity (mean pocket depth, mean attachment loss, and percentage of sites that bled on probing) were found. The diseased group had significantly higher specific IgG responses to the RgpA-Kgp complex than did the control group, and the responses were significantly associated with mean probing depths and percentage of sites positive for P. gingivalis. Analysis of the IgG subclass responses to the RgpA-Kgp complex revealed that the subclass distribution for both the diseased and control groups was IgG4 > IgG2 > IgG3 = IgG1. The IgG2 response to the complex was positively correlated with mean probing depth, whereas the IgG4 response was negatively correlated with this measure of disease severity. Immunoblot analysis of the RgpA-Kgp complex showed that sera from healthy subjects and those with low levels of disease, with high IgG4 and low IgG2 responses, reacted with the RgpA27, Kgp39, and RgpA44 adhesins; however, sera from diseased subjects with low IgG4 and high IgG2 responses reacted only with the RgpA44 and/or Kgp44 adhesins. Epitope mapping of the RgpA27 adhesin localized a major epitope recognized by IgG4 antibodies in sera from subjects with high IgG4 and low IgG2 responses to the RgpA-Kgp complex which was not recognized by sera from diseased subjects with low IgG4 and high IgG2 responses.

FIG. 1

Relationship between the percentage of sites positive for P. gingivalis and mean probing depth (A) and mean attachment loss (B). ●, control group; ▴, diseased group. Linear regression lines are shown.

FIG. 2

Serum IgG and IgM responses to the RgpA-Kgp complex of P. gingivalis. Sera from control subjects (●) and diseased subjects (▴) were used in the ELISA with the RgpA-Kgp complex as the adsorbed antigen. Antibody responses are expressed as the ELISA OD₄₅₀ obtained minus background, with each point representing the mean ± standard deviation of three values.

FIG. 3

Relationship between serum IgG response to the RgpA-Kgp complex and mean probing depth (A) and percentage of sites positive for P. gingivalis (B). ●, control subjects; ▴, diseased subjects. Antibody responses are expressed as the ELISA OD₄₅₀ obtained minus background, with each point representing the mean ± standard deviation of three values.

FIG. 4

Serum IgG subclass responses to the RgpA-Kgp complex of P. gingivalis. Sera from control subjects (●) and diseased subjects (▴) were used in the ELISA with the RgpA-Kgp complex as the adsorbed antigen. Antibody responses are expressed as the ELISA OD₄₅₀ obtained minus background, with each point representing the mean ± standard deviation of three values.

FIG. 5

Relationship between serum IgG2 (A) and IgG4 (B) responses to the RgpA-Kgp complex and disease severity (as mean probing depth). Antibody responses are expressed as the ELISA OD₄₅₀ obtained minus the background, with each point representing the mean ± standard deviation of three values. Only those IgG2 or IgG4 ELISA OD₄₅₀ values which were greater than double the median value of the total IgG response for the control group are shown.

FIG. 6

Immunoblot analysis of responses by human sera from control and diseased patient groups against the RgpA-Kgp complex of P. gingivalis. The transblotted RgpA-Kgp complex after SDS-PAGE was probed with sera from subjects C10 and D24 (low IgG2, high IgG4 response to the RgpA-Kgp complex) and D20 (high IgG2, low IgG4 response to the RgpA-Kgp complex). Molecular mass markers are shown at the right.

FIG. 7

Epitope mapping of the RgpA27 adhesin of the RgpA-Kgp complex. Shown are serum IgG antibody responses, assessed by ELISA, to P. gingivalis W50 RgpA27 overlapping peptides. Twenty-one overlapping pin-bound peptides representing the N-terminal 148 residues of the RgpA27 adhesin were probed with sera from subjects C4 (low IgG response to the RgpA-Kgp complex; ▤), D24 (low IgG2, high IgG4 response to the RgpA-Kgp complex; ▧), C10 (low IgG2, high IgG4 response to the RgpA-Kgp complex; □), and D20 (high IgG2, low IgG4 response to the RgpA-Kgp complex; ■).

FIG. 8

IgG subclass analysis of antibody binding to the overlapping peptides of the RgpA27 adhesin. Twenty-one overlapping peptides representing the N-terminal 148 residues of the RgpA27 adhesin were probed with pooled subject immunoreactive sera. Sera from patients with a positive response to either EP1, EP2, EP3, or EP4 were pooled. The antibody subclass was determined using IgG-specific subclass antibodies IgG1 (□), IgG2 (░⃞), IgG3 (), and IgG4 (■).