Development of fluorescence-based receptor binding assays for the detection

描述(由申请人提供)：有害的藻华(HAB)正在美国海岸外频繁发生，特别是在新英格兰、墨西哥湾和太平洋的沿海水域。从历史上看，赤潮与鱼类死亡和海洋哺乳动物死亡有关；然而，它们对人类健康的影响和海产品受到赤潮污染造成的经济损失正变得越来越普遍，保守估计每年约为8200万美元。赤潮的毒性作用是由不同种类的海藻和微生物产生的毒素沉淀出来的，然后这些毒素被过滤喂养的贝类和鱼类消耗。大量积累可能会导致 对海洋动物和人类的毒性和死亡。海洋浮游植物产生的许多天然毒素是耐热和耐酸的；因此，烹调受污染的海鲜并不能消除中毒的风险。目前，美国各州机构监测有毒浮游植物的存在，当细胞数量达到设定的水平时，对贝类和鱼类进行毒素检测。当毒素水平达到FDA设定的限值时，渔业资源就会关闭。然而，目前在美国检测最常见的与海鲜中毒有关的海洋毒素的方法存在严重缺陷，包括检测时间长、成本高、动物使用、灵敏度和/或样品吞吐量低，或工作范围小。本项目的目的是建立一种基于荧光的受体结合试验(FBA)，用于检测引起麻痹性贝类中毒(萨克毒素)和遗忘性贝类中毒(软骨藻酸)的海洋神经毒素。这些检测方法具有成本低、灵敏度高、动物使用量少等优点，可作为现有方法的快速检测手段。FBA将是对这些毒素的放射配基受体结合分析(RBA)的改进，因为荧光平台不需要使用放射性，因此更安全，而且成本低得多。使用与我们最近针对短杆菌毒素和雪卡毒素开发的FBA相同的技术，荧光团将与岩藻毒素和软骨藻酸结合，并用作标记配体来检测与毒素受体的相互作用。由于RBAs已用于各种样品基质，并被证明与样品中的小鼠生物测定和高效液相色谱结果密切相关，因此，FBAs可能成为检测海鲜或沿海水样中毒素的下一个进步步骤，从而保护人类健康并协助监测渔业资源。

DESCRIPTION (provided by applicant): Harmful algal blooms (HABs) are becoming frequent occurrences off the coasts of the United States, particularly in coastal waters of New England, the Gulf of Mexico, and the Pacific Ocean. Historically, HABs have been associated with fish kills and marine mammal mortalities; however, their effects on human health and economic loss due to HAB contamination of seafood are becoming more prevalent, with conservative estimates around $82 million annually. The toxic effects of HABs are precipitated by the production of toxins by various species of marine algae and microorganisms, which are then consumed by filter feeding shellfish and finfish. Accumulation in significant quantities can result in toxicity and death in marine animals and humans. Many of the natural toxins produced by marine phytoplankton are heat and acid stable; therefore, cooking contaminated seafood does not eliminate the risk of poisoning. Currently, US state agencies monitor for the presence of toxic phytoplankton and when the cell count reaches a set level, shellfish beds and finfish are tested for the presence of toxin. When toxin levels reach FDA set limits, fishery resources are closed. However, current methods for detection of marine toxins most commonly associated with seafood poisonings in the US have serious drawbacks, including lengthy assay time, high cost, animal usage, low sensitivity and/or sample throughput, or small working ranges. The purpose of this project is to develop a fluorescence based receptor binding assay (FBA) for the detection of marine neurotoxins that cause paralytic shellfish poisoning (saxitoxin) and amnesic shellfish poisoning (domoic acid). These assays could be used as a rapid test alternative to current methods and they have the advantage of lower cost, high sensitivity and lower animal usage. The FBA will be an improvement over the radioligand receptor binding assay (RBA) for these toxins, as the fluorescence platform does not require the use of radioactivity and is thus safer and far less expensive. Using the same techniques as the FBA that we recently developed for brevetoxins and ciguatoxins, fluorophores will be conjugated to saxitoxin and domoic acid and used as the labeled ligand to examine interactions with the toxins' receptors. As RBAs have been used for a variety of sample matrices and been shown to strongly correlate with the mouse bioassay and HPLC results in samples, FBAs could be the next progressive step in the detection of toxins in seafood or coastal water samples thereby protecting human health and aiding in the monitoring of fishery resources.