Halter Michael, Almeida Jamie L, Tona Alessandro, Cole Kenneth D, Plant Anne L, Elliott John T
Cell Systems Science Group/Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA.
Assay Drug Dev Technol. 2009 Aug;7(4):356-65. doi: 10.1089/adt.2009.0192.
Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are important for characterizing decontamination strategies and developing detection technologies for field use. We report here an assay for ricin that provides a response that is relevant to the mechanism of ricin activity and permits a much faster readout than the commonly used assays for cytotoxicity. The assay relies on the response of an engineered reporter cell line that was produced by stably transfecting Vero cells to express green fluorescent protein (GFP) under the control ofa cytomegalovirus (CMV) promoter. The results of the GFP-based assay were compared with the assay results from three commercially available cytotoxicity assays. The GFP assay reports a sensitive response to ricin after 6 h of treatment while the other assays require a 24-h incubation. Unlike the other assays, monitoring cellular GFP on a per-cell basis allows detection of reduced ribosome activity before significant cell death occurs, and the results are not convoluted by the numbers of cells being assayed.
用于测量植物毒素蓖麻毒素等蛋白质对核糖体抑制作用的基于细胞的检测方法，对于表征去污策略和开发现场使用的检测技术非常重要。我们在此报告一种针对蓖麻毒素的检测方法，该方法提供了与蓖麻毒素活性机制相关的响应，并且比常用的细胞毒性检测方法能够更快地读出结果。该检测方法依赖于一种经过工程改造的报告细胞系的响应，该细胞系是通过稳定转染非洲绿猴肾细胞（Vero细胞）以在巨细胞病毒（CMV）启动子的控制下表达绿色荧光蛋白（GFP）而产生的。将基于GFP的检测结果与三种市售细胞毒性检测方法的检测结果进行了比较。GFP检测方法在处理6小时后对蓖麻毒素报告出灵敏的响应，而其他检测方法需要24小时的孵育。与其他检测方法不同，基于单个细胞监测细胞GFP能够在显著细胞死亡发生之前检测到核糖体活性降低，并且结果不会因被检测细胞的数量而变得复杂。
