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Hirai, Kimito Department of Pathophysiology—Periodontal Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University
Yamaguchi-Tomikawa, Tomoko Department of Pathophysiology—Periodontal Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University
Eguchi, Toru R&D, Sunstar Inc.
Maeda, Hiroshi Department of Endodontology, Osaka Dental University
Takashiba, Shogo Department of Pathophysiology—Periodontal Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University ORCID Kaken ID publons researchmap
Abstract
Chronic periodontitis is an inflammatory disease caused by the formation of oral microbial biofilms. Periodontitis is associated with general health and not only oral diseases.Porphyromonas gingivalisis a well-known keystone pathogen for periodontitis and is associated with several systemic diseases, such as diabetes mellitus and Alzheimer's disease. We previously developed a system for screening periodontitis usingP. gingivalis-specific serum immunoglobulin G (IgG) in an enzyme-linked immunosorbent assay with a sensitivity of 0.774 and a specificity of 0.586 and an area under the receiver operating characteristic curve of 0.708. However, the antigens elicited non-specific responses, since they were obtained from whole extracts of sonicated cultured bacteria. The purpose of this study was to identify antigens ideal for a sensitive and specific serum test. We identified the specific antigens using immunoaffinity columns immobilized with IgG antibodies from periodontitis patients. Liquid chromatography-tandem mass spectrometry identified 29 antigens from the elutes. Recombinant proteins for these candidates were synthesized using the wheat germ cell-free translation system and screened by dot blot analysis with serum from the columns. Three of the 16 candidates that reacted showed strongest affinities upon dot blot analysis; they included outer membrane protein 28, cysteine proteases, lysine gingipain Kgp, and arginine gingipain RgpA. Outer membrane protein 28 was not suitable for screeningP. gingivalisinfection because of its high false-negative rates. Kgp and RgpA were unstable antigens since they underwent self-digestion. They were made stable by substituting the active cysteine residues in Kgp and RgpA with alanine using site-directed mutagenesis. Using the modified antigens, we demonstrated that the patient serum IgG level against RgpA was the highest among all the antigens expressed inP. gingivalis. Moreover, the N-terminus of recombinant RgpA was excellent in differentiating between diseased and non-diseased states (with sensitivity of 0.85, specificity of 0.9, and area under the curve of 0.915). Although dot blot analysis was the only experiment used, the N-terminus of RgpA is an excellent antigen to immunologically test forP. gingivalisinfection, especially for estimating the risks for periodontitis-associated systemic diseases. In conclusion, we have developed aP. gingivalisantigen for screening periodontitis.
Keywords
screening chronic periodontitis
Porphyromonas gingivalis
serum IgG test
gingipain
specific antigen
Published Date
2020-06-05
Publication Title
Frontiers in Immunology
Volume
volume11
Publisher
Frontiers Media
Start Page
1017
ISSN
1664-3224
Content Type
Journal Article
language
英語
OAI-PMH Set
岡山大学
Copyright Holders
© 2020 Hirai, Yamaguchi-Tomikawa, Eguchi, Maeda and Takashiba.
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publisher
PubMed ID
DOI
Web of Science KeyUT
Related Url
isVersionOf https://doi.org/10.3389/fimmu.2020.01017
License
https://creativecommons.org/licenses/by/4.0/
Funder Name
Ministry of Education, Culture, Sports, Science and Technology
助成番号
22390397