Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy.
J Immunol Methods. 2012 Apr 30;378(1-2):11-9. doi: 10.1016/j.jim.2012.01.011. Epub 2012 Jan 30.
Opsonophagocytosis is the primary mechanism for the clearance of Group B Streptococcus (GBS) by the host, and levels of opsonic antibodies may correlate with protection in preclinical models. A killing-based opsonophagocytosis assay (OPA), can be used to determine the functional activity of vaccine-induced GBS-specific antibodies. The assay, which measures the number of bacterial colonies surviving phagocytic killing in the presence of specific antibodies and complement, is rather expensive, time-consuming and poorly standardized. Here we describe a rapid, sensitive and reproducible fluorescent OPA assay (fOPA) based on flow cytometry analysis (FACS), which allows internalized bacteria to be distinguished from those associated to the plasma membrane of phagocytic cells. Fixed GBS were labeled with pHrodo™, a fluorescent dye which dramatically increases the emitted fluorescence at the acidic conditions present in the phagocytic endosomal compartment. Labeled bacteria were incubated with HL-60 cells differentiated to phagocytes, antibodies and complement, and then analyzed by FACS. A further improvement to our method, allowing to reduce assay variability, consisted on a step of selection of effector cells among the HL-60 population. Analysis of sera from mice immunized with different GBS vaccines revealed comparable sensitivity and specificity with the traditional killing OPA assay (kOPA), and a good correlation between the fluorescent signal of bacteria internalized by HL-60 phagocytes and killing. Remarkably, the pHrodo-based approach reduced the variability observed with other fOPA assays. The obtained data indicate the proposed fOPA as a reliable and useful tool for functional antibody assessment.
调理吞噬作用是宿主清除 B 群链球菌（GBS）的主要机制，调理抗体的水平可能与临床前模型中的保护作用相关。一种基于杀伤的调理吞噬作用测定法（OPA）可用于确定疫苗诱导的 GBS 特异性抗体的功能活性。该测定法测量在存在特异性抗体和补体的情况下吞噬杀伤后存活的细菌菌落数，该测定法非常昂贵、耗时且标准化程度较差。在这里，我们描述了一种基于流式细胞术分析（FACS）的快速、灵敏和可重复的荧光 OPA 测定法（fOPA），该方法允许区分内化的细菌与吞噬细胞质膜相关的细菌。固定的 GBS 用 pHrodo™标记，这是一种荧光染料，在吞噬体腔室中存在的酸性条件下，可大大增加发射的荧光强度。用标记的细菌与分化为吞噬细胞、抗体和补体的 HL-60 细胞孵育，然后通过 FACS 进行分析。我们的方法的进一步改进包括在 HL-60 群体中选择效应细胞的步骤，这可以降低测定变异性。用不同的 GBS 疫苗免疫的小鼠的血清分析显示，与传统的杀伤 OPA 测定法（kOPA）相比，具有相似的灵敏度和特异性，并且 HL-60 吞噬细胞内化的细菌的荧光信号与杀伤之间存在良好的相关性。值得注意的是，基于 pHrodo 的方法降低了与其他 fOPA 测定法观察到的变异性。所得数据表明，所提出的 fOPA 是一种用于功能抗体评估的可靠且有用的工具。
