Development of fluorescence-based receptor binding assays for the detection

开发基于荧光的受体结合检测方法

• 批准号: 8783099
• 负责人: Jennifer Rettew McCall
• 金额: $ 22.44万
• 依托单位: SEATOX RESEARCH, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8783099
• 关键词: Acids; Affinity; Agonist; Algae; Animals; Area; Back; Beds; Binding; Biological Assay; Cell Count; Cessation of life; Ciguatoxins; Color; Cyclophosphamide; Detection; Development; Dissociation; Eating; Ethical Issues; Extinction (Psychology); Fisheries; Fishes; Fluorescence; Geographic state; Goals; Health; Heating; High Pressure Liquid Chromatography; Human; International; Label; Length; Ligands; Mammals; Marine Toxins; Marines; Marketing; Measures; Methods; Mexico; Monitor; Mus; Neurotoxins; New England; Pacific Ocean; Paralysed; Phase; Phytoplankton; Poisoning; Process; Production; Public Health; Radioactive Waste; Radioactivity; Radioligand Assay; Research; Research Personnel; Resources; Risk; Sampling; Sanitation; Savings; Saxitoxin; Seafood; Shellfish; Small Business Technology Transfer Research; Specificity; Techniques; Technology; Testing; Time; Toxic effect; Toxin; Toxin Conjugates; United States; Validation; Work; assay development; authority; base; brevetoxin; coastal water; cooking; cost; domoic acid; feeding; fluorophore; harmful algal blooms; health economics; innovation; killings; microorganism; mortality; novel; programs; prototype; public health relevance; radioligand; receptor; receptor binding; screening; symposium; user-friendly; water sampling

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.

描述（由申请人提供）：有害藻华（HAB）在美国沿海频繁发生，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，HAB 与鱼类死亡和海洋哺乳动物死亡有关。然而，由于海产品 HAB 污染，它们对人类健康和经济损失的影响变得越来越普遍，保守估计每年约为 8200 万美元。 HAB 的毒性作用是由各种海洋藻类和微生物产生的毒素引起的，然后这些毒素被滤食性贝类和有鳍鱼类消耗。大量积累可能会导致 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对热和酸稳定；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，将对贝类床和鱼类进行毒素检测。当毒素水平达到 FDA 设定的限值时，渔业资源就会被关闭。然而，目前在美国检测与海鲜中毒最常见的海洋毒素的方法具有严重的缺点，包括检测时间长、成本高、动物使用、灵敏度和/或样品通量低或工作范围小。该项目的目的是开发一种基于荧光的受体结合测定（FBA），用于检测导致麻痹性贝类中毒（石房蛤毒素）和遗忘性贝类中毒（软骨藻酸）的海洋神经毒素。这些测定可用作当前方法的快速测试替代方案，并且具有成本较低、灵敏度高和动物使用量较低的优点。 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，因此更安全且便宜得多。使用与我们最近为短尾毒素和雪卡毒素开发的 FBA 相同的技术，荧光团将与石房蛤毒素和软骨藻酸缀合，并用作标记配体来检查与毒素受体的相互作用。由于 RBA 已用于各种样品基质，并且已被证明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中毒素的下一个进步步骤，从而保护人类健康和协助监测渔业资源。