Detection of antigenic proteins in Porphyromonas gingivalis using two-dimensional electrophoresis and western blots.
Using two-dimensional (2D) electrophoresis and western blot assay, we analyzed antigenic proteins in Porphyromonas gingivalis uniquely recognized by antibodies in sera of periodontitis subjects. Proteins in the total membrane fraction of P. gingivalis 381 were resolved into at least 70 protein spots by 2D electrophoresis. In the gel stained with silver, the substance around 47 kDa protein on the acidic side (at an isoelectric point of about 4.5) was stained as a smear. Antigenic substances were characterized using purified IgGs from sera of 16 adult periodontitis (AP), 19 rapidly progressive periodontitis (RPP) and 14 periodontally healthy volunteers. Western blots demonstrated that 75 kDa protein reacted with IgGs from 75% of AP patients (p < 0.001), the antigenic substance around acidic 47 kDa protein reacted with IgGs from 81.3% of AP (p < 0.01) and 68.4% of RPP patients (p < 0.01) and the acidic 47 kDa protein reacted with 87.5% of AP (p < 0.01) and 78.9% of RPP patients (p < 0.01). The reaction frequency was significantly different from that of the healthy volunteers. Also 51 kDa and 41 kDa proteins reacted with 47 and 43 of 49 IgG samples, respectively. The substance around acidic 47 kDa protein reacted with mouse antiserum to P. gingivalis-LPS. After treatment with pronase or heat, the antigenic reactions disappeared not only from the proteins, but also from the area around the acidic 47 kDa protein. When the fraction was digested with lipase, the antigenic reaction of the area decreased.(ABSTRACT TRUNCATED AT 250 WORDS)
D000328:Adult; D000368:Aged; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D004731:Endotoxins / Q000737:chemistry* / Q000302:isolation & purification; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D008050:Lipid A / Q000737:chemistry* / Q000302:isolation & purification; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry; D008875:Middle Aged; D008970:Molecular Weight; D010518:Periodontitis / Q000097:blood / Q000276:immunology / Q000382:microbiology*; D016966:Porphyromonas gingivalis / Q000276:immunology*; D018709:Statistics, Nonparametric
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004731:Endotoxins; D007074:Immunoglobulin G; D008050:Lipid A; D008565:Membrane Proteins; C027315:lipid A-associated protein, Bacteria