Ahn-Yoon Soohyoun, DeCory Thomas R, Baeumner Antje J, Durst Richard A
Department of Food Science & Technology, Cornell University, Geneva, New York 14456-0462, USA.
Anal Chem. 2003 May 15;75(10):2256-61. doi: 10.1021/ac026428t.
An extremely sensitive bioassay has been developed for cholera toxin (CT) detection, using ganglioside-incorporated liposomes. Cholera is a diarrheal disease, often associated with water or seafood contamination. Ganglioside GM1 was used to prepare the liposomes by spontaneous insertion into the phospholipid bilayer. CT recognition and signal generation is based on the strong and specific interaction between GM1 and CT. In a sandwich immunoassay, CT was detected as a colored band on the nitrocellulose membrane strip, where CT bound to GM1-liposomes can be captured by immobilized antibodies. The intensity of the band could be visually estimated or measured by densitometry, using computer software. The limit of detection (LOD) of CT in the assay system was found to be 10 fg/mL which is equivalent to 8 zmol in the 70-microL sample. The assay was also tested with water samples spiked with CT, providing a LOD of 0.1-30 pg/mL, which is much better than previously reported limits of detection from other assays. The assay could be completed within 20 min. These results demonstrate that the bioassay developed for CT is rapid and ultrasensitive, suggesting the possibility for detecting CT, simply and reliably, in field screening.
已开发出一种极其灵敏的生物测定法用于霍乱毒素（CT）检测，该方法使用掺入神经节苷脂的脂质体。霍乱是一种腹泻病，常与水或海产品污染有关。神经节苷脂GM1通过自发插入磷脂双分子层来制备脂质体。CT的识别和信号产生基于GM1与CT之间强烈且特异的相互作用。在夹心免疫测定中，CT在硝酸纤维素膜条上被检测为一条有色带，其中与GM1 - 脂质体结合的CT可被固定化抗体捕获。该条带的强度可通过目视估计或使用计算机软件通过光密度测定法进行测量。测定系统中CT的检测限（LOD）为10 fg/mL，在70微升样品中相当于8 zmol。还用添加了CT的水样对该测定法进行了测试，其检测限为0.1 - 30 pg/mL，远优于先前报道的其他测定法的检测限。该测定可在20分钟内完成。这些结果表明，所开发的用于CT的生物测定法快速且超灵敏，这表明在现场筛查中能够简单可靠地检测CT的可能性。
