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

描述(由申请人提供)：为了满足FDA对制造产品中肉毒杆菌神经元毒素A(BoNT/A)的可靠评估的要求，我们建议应用荧光肽信标技术在一次检测中同时测量毒素的结合功能和蛋白分解功能，从而实现“从实验室到市场”。由于这是一项同质技术，它可以修改为易于使用、可移植、自动化和高吞吐量。在这里，我们建议开发一系列新的荧光试剂，将它们与固定化抗体在一个独立的系统中结合使用，以实现对复杂样品中BONT/A的可靠和高灵敏度的检测。具体来说，我们的目标如下：特定目标#1：设计、合成和测试新型荧光肽信标作为检测毒素结合功能的示踪剂；特定目标#2：设计、合成和测试新型荧光肽以检测毒素的蛋白分解功能；特定目标#3：设计、合成和测试由上述两种多肽通过柔性接头连接而成的新型融合肽，以检测毒素的两种功能。具体目标#4：设计和开发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。具体目标#5：修改现有的便携式荧光读取器/孵化器，将其用作测量荧光动力学的试验台，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的工作经验，我们相信拟议的概念将被证明是可行的。我们希望拟议的工作将为将该项目启动到下一开发阶段奠定坚实的基础，在这一阶段，我们将建立体外测试结果与使用复杂样本矩阵的小鼠生物检测的相关性，并开发稳健的仪器。我们的长期目标是寻求FDA的批准，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、边境管制等的常规部署。 公共卫生相关性：肉毒杆菌神经元毒素(BoNTs)是一类由肉毒梭菌产生的强效神经元毒素，广泛存在于自然界。虽然这些毒素可以在意外感染、食物中毒或生物攻击事件中导致疾病和人员伤亡，但它们也被广泛用于治愈疾病和提高超过75个国家数百万人的生活质量。因此，我们建议开发新的试剂和检测系统，以准确地测量制成品中毒素的效力，以帮助防止过量或不足剂量。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、边境管制等领域。

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.