Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Development and Optimization of a Rapid Colorimetric Membrane Immunoassay for Porphyromonas gingivalis

  • Lee, Jiyon (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Choi, Myoung-Kwon (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kim, Jinju (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Chun, SeChul (Department of Environmental Health Science, Konkuk University) ;
  • Kim, Hong-Gyum (STARGO Inc.) ;
  • Lee, HoSung (STARGO Inc.) ;
  • Kim, JinSoo (STARGO Inc.) ;
  • Lee, Dongwook (Department of Oral Microbiology and Immunology, DRI and BK21 Plus Program, School of Dentistry, Seoul National University) ;
  • Han, Seung-Hyun (Department of Oral Microbiology and Immunology, DRI and BK21 Plus Program, School of Dentistry, Seoul National University) ;
  • Yoon, Do-Young (Department of Bioscience and Biotechnology, Konkuk University)
  • Received : 2021.03.17
  • Accepted : 2021.04.02
  • Published : 2021.05.28
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Abstract

Porphyromonas gingivalis (P. gingivalis) is a major bacterial pathogen that causes periodontitis, a chronic inflammatory disease of tissues around the teeth. Periodontitis is known to be related to other diseases, such as oral cancer, Alzheimer's disease, and rheumatism. Thus, a precise and sensitive test to detect P. gingivalis is necessary for the early diagnosis of periodontitis. The objective of this study was to optimize a rapid visual detection system for P. gingivalis. First, we performed a visual membrane immunoassay using 3,3',5,5'-tetramethylbenzidine (TMB; blue) and coating and detection antibodies that could bind to the host laboratory strain, ATCC 33277. Antibodies against the P. gingivalis surface adhesion molecules RgpB (arginine proteinase) and Kgp (lysine proteinase) were determined to be the most specific coating and detection antibodies, respectively. Using these two selected antibodies, the streptavidin-horseradish peroxidase (HRP) reaction was performed using a nitrocellulose membrane and visualized with a detection range of 103-105 bacterial cells/ml following incubation for 15 min. These selected conditions were applied to test other oral bacteria, and the results showed that P. gingivalis could be detected without cross-reactivity to other bacteria, including Streptococcus mutans and Escherichia fergusonii. Furthermore, three clinical strains of P. gingivalis, KCOM 2880, KCOM 2803, and KCOM 3190, were also recognized using this optimized enzyme immunoassay (EIA) system. To conclude, we established optimized conditions for P. gingivalis detection with specificity, accuracy, and sensitivity. These results could be utilized to manufacture economical and rapid detection kits for P. gingivalis.

Keywords

Acknowledgement

This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (2021R1A2C 3014577) and was written as part of the Konkuk University Researcher Fund in 2020.

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