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Flow‐Cytometric Identification and Detection of Porphyromonas gingivalis by a LPS Specific Monoclonal Antibody
Author(s) -
Kamiya Ichiro,
Okuda Kazuhiro,
Hara Kohji
Publication year - 1994
Publication title -
journal of periodontology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.036
H-Index - 156
eISSN - 1943-3670
pISSN - 0022-3492
DOI - 10.1902/jop.1994.65.4.309
Subject(s) - porphyromonas gingivalis , fusobacterium nucleatum , prevotella intermedia , microbiology and biotechnology , monoclonal antibody , bacteroides , bacteroidaceae , chemistry , bacteria , actinobacillus , flow cytometry , antibody , biology , immunology , genetics
T he purpose of this study was to identify Porphyromonas gingivalis (P. gingivalis) by flow cytometry (FCM) using a monoclonal antibody (MAb) OMR‐Bg1E directed to P. gingivalis ‐specific lipopolysaccharide (LPS). The P. gingivalis strains ATCC 33277, 381, ES075, W50, and A7A1 were selected for the study. Fusobacterium nucleatum (F. nucleatum), Prevotella intermedia (P. intermedia), Campylobacter rectus (C. rectus), Streptococcus sanguis (S. sanguis) and Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) served as controls. A suspension of 10 7 bacteria/ml of each bacteria was prepared and then reacted with a P. gingivalis specific MAb OMR‐Bg1E and fluorescein isothiocyanate (FITC) labeled second antibody. These samples were analyzed by FCM. Bacterial specific binding aggregate on data was separated out by the forward and side‐angle‐scatter characteristics, while non‐specific binding (NSB) was eliminated by excluding the region with mouse IgG‐positive and second antibody‐positive area. FCM detected a mean range of 56.2% to 97.2% P. gingivalis strains. There was a 5.1% non‐specific binding using FCM to non‐ P. gingivalis strains. When the P. gingivalis concentration was adjusted to 10 2 , 10 4 , and 10 6 bacteria/ml, a detection rate of 35.7%, 48.1%, and 91.4%, was respectively observed. The lower sensitivity of the flow cytometric assay was 10 2 bacteria/ml. When P. gingivalis was added to P. intermedia suspension at 1, 20, 40, 60, and 80%, the MAb‐positive fraction yielded by FCM displayed a coefficient of determination of 0.967 with the actual percentage of P. gingivalis and could be regressed to a linear function. The same P. gingivalis strain was then added to P. gingivalis ‐fite native plaque samples at a final concentration of 0, 1, 5, 10, 15, 20, 40, 60, and 80%. The detection rate by flow cytometric analysis showed a significant correlation ( P < 0.0001) with the actual percentage of P. gingivalis in the spiked plaque sample and the coefficient of determination to a linear regression curve was as high as 0.983. In competition experiments incubating MAb with LPS purified from P. gingivalis , subsequent reaction of MAb with the P. gingivalis whole cell was reduced. This study demonstrates the reliability of FCM analysis of P. gingivalis by the use of the MAb OMR‐Bg1E. Flow‐cytometric identification using P. gingivalis or other bacteria‐specific MAbs could offer a means for rapid screening and semi‐quantitation of periodontopathic bacteria in mixed cultures or plaque samples. J Periodontol 1994; 65:309–315 .