Flow-cytometric identification and detection of Porphyromonas gingivalis by a LPS specific monoclonal antibody.
The 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, ESO75, 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Kamiya
I
I
Department of Periodontology, Niigata University School of Dentistry, Japan.
Okuda
K
K
Hara
K
K
D000704:Analysis of Variance; D000911:Antibodies, Monoclonal; D015169:Colony Count, Microbial / Q000379:methods*; D016001:Confidence Intervals; D003773:Dental Plaque / Q000382:microbiology*; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D016014:Linear Models; D008070:Lipopolysaccharides / Q000276:immunology*; D016966:Porphyromonas gingivalis / Q000276:immunology / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D008070:Lipopolysaccharides; D016650:Fluorescein-5-isothiocyanate