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）：有害的藻华（HABS）正经常出现在美国沿海，尤其是在新英格兰沿海水域，墨西哥湾和太平洋的沿海水域。从历史上看，哈布斯与鱼类杀和海洋哺乳动物的死亡率有关。但是，它们对海鲜污染海鲜造成的人类健康和经济损失的影响越来越普遍，保守的估计每年约8200万美元。 HAB的毒性作用是由各种海洋藻类和微生物的毒素产生引起的，然后通过过滤喂食贝类和鳍鱼会消耗它们。大量积累可能会导致 在海洋动物和人类中的毒性和死亡中。海洋浮游植物产生的许多天然毒素都是热和酸稳定的。因此，烹饪污染的海鲜不会消除中毒的风险。当前，美国州机构监视着有毒浮游植物的存在，当细胞计数达到设定水平时，贝类床和鳍鱼将被测试是否存在毒素。当毒素水平达到FDA设定限制时，渔业资源就会关闭。但是，目前检测美国最常见的海鲜毒素的方法在美国具有严重的缺点，包括冗长的测定时间，高成本，动物使用，低灵敏度和/或样品吞吐量或小型工作范围。该项目的目的是开发基于荧光的受体结合测定法（FBA），以检测导致麻痹贝类中毒（saxitoxin）和健忘症贝类中毒（Domoic Acid）的海洋神经毒素。这些测定可以用作当前方法的快速测试替代方法，它们具有较低的成本，高灵敏度和较低的动物使用情况。对于这些毒素，FBA将是对放射性配体受体结合测定法（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Brevetoxins和Ciguatoxins开发的FBA相同的技术，荧光团将与萨克西毒素和多糖酸结合，并用作标记的配体来检查与毒素受体的相互作用。由于RBA已用于多种样品矩阵，并被证明与小鼠生物测定有密切相关，而HPLC在样品中导致了样品，因此FBA可能是检测海鲜或沿海水样品中毒素的下一个渐进步骤，从而保护人类健康并有助于监测渔业资源。