Novel Method and Self Contained System for Reliable Assessment of Potency of Botu

可靠评估博土功效的新方法和独立系统

• 批准号: 8126652
• 负责人: James N Herron
• 金额: $ 34.2万
• 依托单位: AGRI-ANALYSIS, LLC
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8126652
• 关键词: Antibodies; Area; Automation; Bacteria; Binding; Biological; Biological Assay; Botulinum Toxins; C-terminal; Cleaved cell; Clostridium botulinum; Complex; Cosmetics; Country; Detection; Development; Devices; Disease; Dose; Epitopes; Event; Fluorescence; Fluorogenic Substrate; Food Poisoning; Foundations; Goals; Hospitals; Immobilization; Incubators; Infection; Kinetics; Label; Light; Link; Marketing; Measurement; Measures; Membrane; Methods; Mus; Nature; Neurons; One-Step dentin bonding system; Peptides; Pharmacologic Substance; Plants; Pyroxylin; Reader; Reagent; Reporter; Sampling; Screening procedure; Series; Signal Transduction; Staging; Surface; System; Technology; Test Result; Testing; Time; Toxin; Tracer; Work; World Health Organization; assay development; base; botulinum; design; experience; flexibility; improved; in vitro testing; instrumentation; meetings; novel; portability; presynaptic; prevent; protein aminoacid sequence; sound; synaptosomal-associated protein 25

DESCRIPTION (provided by applicant): To meet Agency requirement for reliable assessment of botulinum neuron toxin A (BoNT/A) in manufactured products, we propose to apply the fluorescent peptide beacon technology for measurement of both binding function and proteolytic function of the toxin in a single assay, hence "from lab to marketplace". Because this is a homogenous technology, it is amendable to ease of use, portability, automation and high throughput. Here we propose to develop a series of novel fluorescent reagents, to use them in conjunction with immobilized antibodies in a self- contained system to achieve robust and high sensitivity detection of BoNT/A in complex samples. Specifically, our objectives are as follows: Specific Aim #1: Design, synthesis and testing of a novel fluorescent peptide beacon as tracer to detect the binding function of the toxin; Specific Aim #2: Design, synthesis and testing of a novel fluorogenic peptide to detect the proteolytic function of the toxin; Specific Aim #3: Design, synthesis and testing of a novel fusion peptide made of the above two peptides linked together via flexible linker to detect both functions of the toxin. Specific Aim #4: Design and develop a surface immobilization strategy and self-contained sample handling system to increase sensitivity and ease of use. Specific Aim #5: Modify an existing portable fluorescent reader/incubator to use as testbed for measuring fluorescence kinetics, and correlate such measurement with potency units. Based on our experience in working on these types of systems, we feel confident the proposed concepts would be demonstrated feasible. We hope the proposed work would lay a sound foundation to launch this project to the next stage of development, where we would establish correlation of the in vitro testing results with mouse bioassays using complex sample matrices and on developing robust instrumentation. Our long term goal is to seek FDA approval to manufacture and market this method and device for routine deployment in manufacturing plant, regulatory agencies, world health organizations, hospitals and doctor's office, border control, etc. PUBLIC HEALTH RELEVANCE: Botulinum Neuron Toxins (BoNTs) are a class of potent neuron toxins produced by Clostridium botulinum bacterium widely found in nature. While these toxins can cause diseases and casualties in events of accidental infection, food poisoning, or biological attack, they are also widely used to cure diseases and improve the quality of lives for millions of people in over 75 countries. We hereby propose to develop novel reagents and assay system to accurately measure the potency of the toxins in manufactured products to help prevent over- or under- dose. When fully developed, the system could be deployed in manufacturing plant, regulatory agencies, hospitals, and doctor's offices, world health organizations, border control, etc.

描述（由申请人提供）：为了满足制造产品中肉毒神经元毒素A（BONT/A）可靠评估的代理要求，我们建议应用荧光肽信标技术，以测量单个分析中毒素的结合功能和蛋白水解功能，从而从实验室到市场”。因为这是一项同质技术，因此可以易于使用，可移植性，自动化和高通量。在这里，我们建议开发一系列新型的荧光试剂，并将它们与自动包含系统中的固定抗体结合使用，以实现复杂样品中BONT/A的稳健敏感性检测。具体而言，我们的目标如下：特定目的＃1：新型荧光肽信标的设计，合成和测试是检测毒素的结合功能的示踪剂；特定目标＃2：新型荧光肽的设计，合成和测试以检测毒素的蛋白水解功能；特定目的＃3：设计，合成和测试的新型融合肽由上述两个肽通过柔性接头连接在一起，以检测毒素的两个功能。特定目的＃4：设计和制定表面固定策略和独立的样品处理系统，以提高灵敏度和易用性。特定目标＃5：修改现有的便携式荧光读取器/孵化器作为测试床，用于测量荧光动力学，并将这种测量与效力单位相关联。根据我们在这些类型系统上工作的经验，我们认为可以证明提出的概念可行。我们希望拟议的工作将为下一个开发阶段启动该项目奠定合理的基础，在那里我们将使用复杂的样品矩阵和开发强大的仪器建立体外测试结果与鼠标生物测定结果的相关性。我们的长期目标是寻求FDA批准，以制造和销售这种方法和设备，用于制造工厂，监管机构，世界卫生组织，医院和医生办公室，边境控制等方面的常规部署。 公共卫生相关性：肉毒杆菌神经元毒素（BONTS）是一类有效的神经元毒素，该神经元毒素是由肉毒梭菌细菌在自然界广泛发现的。尽管这些毒素会在意外感染，食物中毒或生物学攻击的事件中引起疾病​​和伤亡，但它们也被广泛用于治愈疾病并改善75个以上国家的数百万人的生活质量。我们在此提议开发新型试剂和测定系统，以准确测量制成品中毒素的效力，以帮助防止过度或不足。完全开发后，该系统可以部署在制造工厂，监管机构，医院和医生办公室，世界卫生组织，边境控制等。