FLOW CYTOMETRIC ANALYSIS OF ORAL ACTINOBACILLUS

口腔放线杆菌的流式细胞术分析

• 批准号: 3447200
• 负责人: William Buchanan
• 金额: $ 5.15万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-03-01 至 1989-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3447200
• 关键词: Actinobacillus; cell population study; dental disorder diagnosis; flow cytometry; immunofluorescence technique; monoclonal antibody; oral bacteria; periodontium disorder

Studies show that species-specific immunofluorescent antibody labeling of bacteria provides an alternative method to complex anaerobic culturing for the quantitative identification specific periodontopathogenic bacteria. Analysis of bacterial fluorescent antibody labeling with fluorescent microscopy is laborious and yields subjective data on fluorescence intensity for only several hundred cells in each sample. Flow cytometers measure individual cell fluorescence as cells flow past a laser with sensitive optics and electronics at a rate of thousands of cells per second. Recent reports suggest that flow cytometry provides an alternative way to measure bacterial cell fluorescence of large numbers of bacteria. The use of lasers to excite fluorescence and sensitive photomultiplier tubes to measure fluorescence by flow cytometers permits use of lower titers of labeling antibody which results in a reduction of non-specific labeling. This study will use monoclonal antibodies that label all tested strains of Actinobacillus actinomycetemcomitans (Aa) to define an immunofluorescent labeling protocol which optimizes flow cytometric detection of Aa cells within bacterial cell mixtures. Indirect and direct immunofluorescent labeling techniques will be compared using FITC conjugated antibodies. The sensitivity and specificity of flow cytometric analyses using optimized flow cytometric technique for detecting Aa cells within mixtures will be compared to analysis of the same mixtures using fluorescence microscopy. Effects that controlled variables in flow cytometric analysis techniques or in bacteria cells have on measuring Aa cells within mixtures will be defined. Bacteria cell variables to be studied include cell preservation techniques, cell concentration or size, and cell growth cycle. Use of nucleic acid staining and addition of an internal bead standard will be tested to see if either improves analyses. Finally, flow cytometric analyses will be used to assess alterations in Aa cell morphology, DNA or protein content produced by in-vitro antibiotic exposure. These cell alterations will be compared to changes in numbers of colony forming units. The techniques defined by this study will provide new investigative tools to measure Aa infection in larger populations.

研究表明，种特异性免疫荧光抗体标记 细菌提供了复杂厌氧培养的替代方法， 定量鉴定特定牙周致病菌。 荧光标记细菌荧光抗体的分析 显微镜是费力的，并且产生关于荧光的主观数据 每个样品中仅几百个细胞的强度。 流式细胞仪 当细胞流过激光器时测量单个细胞荧光， 灵敏的光学器件和电子器件， 一下 最近的报告表明，流式细胞术提供了一种替代方法， 一种测量大量细菌的细菌细胞荧光的方法。 利用激光激发荧光和灵敏的光电倍增管 通过流式细胞仪测量荧光的试管允许使用较低的 标记抗体的滴度，其导致非特异性抗体的减少。 标签。 这项研究将使用标记所有测试的单克隆抗体 放线共生放线杆菌（Actinobacillusactinomycetemcomitans，Aa）菌株，以定义 优化流式细胞术的免疫荧光标记方案 检测细菌细胞混合物中的Aa细胞。 间接和直接 将使用FITC比较免疫荧光标记技术 结合抗体。 流式细胞仪检测的敏感性和特异性 使用优化的流式细胞术技术检测Aa细胞的分析 混合物内的分析将与使用 荧光显微镜 流量控制变量的影响 细胞计数分析技术或在细菌细胞中测定Aa 将定义混合物内的细胞。 细菌细胞变量是 研究包括细胞保存技术，细胞浓度或大小， 和细胞生长周期。 核酸染色的使用和添加核酸染色剂的方法 将测试内部珠标准品以观察是否改善分析。 最后，流式细胞术分析将用于评估Aa的改变 体外抗生素产生的细胞形态、DNA或蛋白质含量 exposure. 这些细胞的变化将与细胞数量的变化进行比较。 菌落形成单位 本研究所定义的技术将提供 新的调查工具，以衡量Aa感染在更大的人群。