基于单克隆抗体的多元弧菌流式细胞术检测技术的研究

A flow cytometry method for detecting multi-vibrios using anti-OmpW monoclonal antibody

摘要:

【目的】建立一种基于单克隆抗体的多元弧菌流式细胞仪检测技术。【方法】以副溶血弧菌表面蛋白r-OmpW的单克隆抗体(mAb)为基础, 以细胞染色率为指标优化流式细胞仪检测副溶血弧菌时所需mAb的反应浓度和反应时间。通过菌落数对比评价在优化条件下流式细胞术方法的准确度、检出限和精密度。根据所建立的流式细胞术平台分析鉴定单抗对其它5种多元弧菌的特异性。【结果】流式细胞仪检测副溶血弧菌时所需r-OmpW单克隆抗体的优化反应浓度为20 mg/L, 反应时间为60 min。当菌浓在104?107cells /mL范围时, 检测值可信度较高, 可特异性识别5种病原弧菌。对含不同菌体浓度的样品进行重复检测, 变异系数均在7%以内。【结论】所建立的这种基于单克隆抗体的多元弧菌流式细胞仪检测技术可快速准确地检测多种病原弧菌。

关键词:单克隆抗体, 多元弧菌, 流式细胞术

Abstract:

[Objective] To establish the flow cytometry (FCM) method for detecting multi-vibrio based on anti-OmpW monoclonal antibody. [Methods] With the rate of cell stainning as an indicator, optimize the reactived conditions of anti-OmpW mAb concentration and reaction time for detecting Vibrio parahaemolyticus by flow cytometry. Evaluate the accuracy, detection limits and precision of flow cytometry by comparing the munber of bacteria calculated by FCM with that in the culture. Based on the obove flow cytometry method, analysize and identify the mAb specificity to the five vibrios. [Results] The optimized mAb concentraion was 20 mg/L, and the optimized reaction time of mAb was 60min when Vibrio parahaemolyticus was detected by flow cytometry. The FCM method could provide high reliability which could specifically recognize the five vibrios in the appropriate cell concentration of 104?107cells/mL. To detect different cell concentration samples repeatedly, the coefficient of variation was less than 7%. [Conclusion] The established flow cytometry method based on anti-OmpW mAb could quickly and accurately detect multiple vibrios.

Key words:Monoclonal antibody, multi-Vibrio, flow cytometry