Development of fluorescence-based receptor binding assays for the detection

描述（由申请人提供）：有害藻华（HAB）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沃茨。从历史上看，有害藻华与鱼类死亡和海洋哺乳动物死亡有关;然而，由于有害藻华污染海产品，其对人类健康的影响和经济损失正变得越来越普遍，保守估计每年约为8 200万美元。各种海洋藻类和微生物产生毒素，然后被滤食性贝类和有鳍鱼类消耗，从而加速了有害藻华的毒性效应。大量积累会导致 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热和酸稳定的;因此，烹饪受污染的海鲜并不能消除中毒的风险。目前，美国国家机构监测有毒浮游植物的存在，当细胞计数达到设定水平时，贝类床和有鳍鱼被检测毒素的存在。当毒素水平达到FDA规定的限度时，渔业资源将被关闭。然而，目前用于检测海洋毒素的方法最常见的与海鲜中毒在美国有严重的缺点，包括冗长的测定时间，高成本，动物的使用，低灵敏度和/或样品通量，或小的工作范围。本项目旨在建立一种基于荧光的受体结合分析法（FBA），用于检测麻痹性贝类毒素（石房蛤毒素）和失忆性贝类毒素（软骨藻酸）。该方法具有成本低、灵敏度高、动物用量少等优点，可作为目前检测方法的快速替代方法。FBA将是对这些毒素的放射性配体受体结合测定（RBA）的改进，因为荧光平台不需要使用放射性，因此更安全，更便宜。使用与我们最近开发的用于短毒素和雪卡毒素的FBA相同的技术，荧光团将与石房蛤毒素和软骨藻酸缀合，并用作标记配体以检查与毒素受体的相互作用。由于RBA已用于各种样品基质，并显示与样品中的小鼠生物测定和HPLC结果密切相关，因此FBA可能是检测海鲜或沿海水样品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.