Studies on Antigenic Proteins of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans by Western Blotting Assay Using Purified IgGs from Patients with Periodontitis

Abstract

This study was designed to investigate antigenic proteins from Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans using purified IgGs from patients with periodontitis. Each bacterial cell was ultrasonically disrupted. Sonicated extracts were divided into three cell fractions by centrifugation: membrane fraction, ribosomal fraction, and S-100 fraction. The membrane fraction was initially divided by centrifugation (25, 000. g for 30min.) into precipitates. The supernatants were split into ribosomal and S-100 fractions by ultracentrifugation (100, 000. g for 2hours). In addition, each bacterial species was grown in diffusate broth medium. After the bacterial cells had been removed, culture supernatants were prepared as culture extract fractions. These four fractions were analyzed by SDS-PAGE and Western blotting assay. Antigenic characterizations were performed using purified IgGs from 8 healthy subjects, 9 patients with adult periodontitis and 9 with rapidly progressive periodontitis. Western blotting assay demonstrated that 47 kDa and 44 kDa proteins from the membrane fraction of P. gingivalis, a 64 kDa protein from the ribosomal fraction of A. actinomycetemcomitans, and a 35 kDa protein from the culture extract fraction of A. actinomycetemcomitans reacted with more than half of the IgGs from the periodontitis cases. In contrast, less than half reacted with the IgGs of healthy subjects. There were no antigenic proteins which reacted exclusively with IgGs from subjects with adult periodontitis or rapidly progressive periodontitis.