Van Dolah F M, Finley E L, Haynes B L, Doucette G J, Moeller P D, Ramsdell J S
Marine Biotoxins Program, Charleston Laboratory, U.S. National Marine Fisheries Service, SC 29412.
Nat Toxins. 1994;2(4):189-96. doi: 10.1002/nt.2620020407.
The lack of rapid, high throughput assays is a major obstacle to many aspects of research on marine phycotoxins. Here we describe the application of microplate scintillation technology to develop high throughput assays for several classes of marine phycotoxin based on their differential pharmacologic actions. High throughput "drug discovery" format microplate receptor binding assays developed for brevetoxins/ciguatoxins and for domoic acid are described. Analysis for brevetoxins/ciguatoxins is carried out by binding competition with [3H] PbTx-3 for site 5 on the voltage dependent sodium channel in rat brain synaptosomes. Analysis of domoic acid is based on binding competition with [3H] kainic acid for the kainate/quisqualate glutamate receptor using frog brain synaptosomes. In addition, a high throughput microplate 45Ca flux assay for determination of maitotoxins is described. These microplate assays can be completed within 3 hours, have sensitivities of less than 1 ng, and can analyze dozens of samples simultaneously. The assays have been demonstrated to be useful for assessing algal toxicity and for assay-guided purification of toxins, and are applicable to the detection of biotoxins in seafood.
缺乏快速、高通量的检测方法是海洋藻毒素研究诸多方面的主要障碍。在此，我们描述了微孔板闪烁技术的应用，基于几类海洋藻毒素的不同药理作用开发高通量检测方法。文中介绍了为短裸甲藻毒素/雪卡毒素和软骨藻酸开发的高通量“药物发现”形式的微孔板受体结合检测方法。对短裸甲藻毒素/雪卡毒素的分析是通过与[3H] PbTx - 3竞争结合大鼠脑突触体电压依赖性钠通道上的位点5来进行的。软骨藻酸的分析基于与[3H] 海藻酸竞争结合蛙脑突触体上的海藻酸/quisqualate谷氨酸受体。此外，还描述了一种用于测定刺尾鱼毒素的高通量微孔板45Ca通量检测方法。这些微孔板检测可在3小时内完成，灵敏度低于1纳克，且能同时分析数十个样品。这些检测方法已被证明可用于评估藻类毒性和毒素的检测导向纯化，适用于检测海产品中的生物毒素。
