Flow‐Cytometric Identification and Detection of Porphyromonas gingivalis by a LPS Specific Monoclonal Antibody

Abstract

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, 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 107 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 102, 104, and 106 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 102 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.

Methods of detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis in periodontal microbiology, with special emphasis on advanced molecular techniques: a review

“…Cytofluorography or flow cytometry for the rapid identification of oral bacteria involves labelling bacterial cells from a patient plaque sample with both species-specific antibodies and a second fluorescein-conjugated antibody. The suspension is then introduced into the flow cytometer, which separates the bacterial cells into an almost single-cell suspension by means of a laminar flow through a narrow tube (Kamiya et al 1994). The sophistication and cost involved in this procedure precludes its wide usage ELISA is similar in principle to other radioimmunoassays, but instead of the radioisotope, an enzymatically derived colour reaction is substituted as the label.…”

Flow cytometry for assessing biocompatibility

“…Thus, the major feature of FCM, that measurements are made separately for each individual cell in the population rather than providing only an average value for all cells in the population, has enabled the technique to be used accurately and simultaneously to determine multiple parameters of cell phenotype and function in a number of diverse areas, including cell-cycle analysis, 7 lymphocyte subsets in immunologically based diseases, 8 tissue-specific fibroblasts, 9 and microbial biology. 10 Moreover, in our laboratory, we developed a procedure for using laser-based FCM to measure intracellular as well as cell-surface antigens, and showed that the relative levels of each of the antigens, measured by arbitrary fluorescence units, is proportional to the actual number of molecules present in the cell. 11 We also used this technique to examine basic cellular and molecular features of muscle differentiation 12 and disease-specific stromal cell subpopulations.…”

Cytotoxicity and altered c‐myc gene expression by medical polyacrylamide hydrogel

“…10 -12 The ability to determine multiple parameters of cell phenotype and function has consequently been utilized in a wide range of applications. [13][14][15] To explore the mechanism of the toxicity of PAMG, FCM was used to observe the effects of PAMG on human fibroblasts in detail.…”