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对制成品中肉毒杆菌神经元毒素A （BoNT/A）可靠评估的要求，我们建议应用荧光肽信标技术在一次分析中测量毒素的结合功能和蛋白水解功能，从而“从实验室到市场”。由于这是一种同质技术，因此它易于使用，可移植性，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，将它们与固定抗体结合在一个独立的系统中，以实现复杂样品中BoNT/ a的鲁棒性和高灵敏度检测。具体而言，我们的目标如下：具体目标#1：设计、合成和测试一种新型荧光肽信标作为示踪剂，以检测毒素的结合功能；特定目标#2：设计、合成和测试一种新型荧光肽，以检测毒素的蛋白水解功能；具体目标#3：设计、合成和测试一种新型融合肽，该融合肽由上述两种肽通过柔性连接物连接在一起，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/培养箱，用作测量荧光动力学的测试平台，并将这种测量与效价单位相关联。根据我们在这些类型的系统上的工作经验，我们有信心提出的概念将被证明是可行的。我们希望所提出的工作将为启动该项目进入下一阶段的开发奠定坚实的基础，在那里我们将建立体外测试结果与使用复杂样品基质的小鼠生物测定的相关性，并开发强大的仪器。我们的长期目标是寻求FDA批准生产和销售这种方法和设备，用于制造厂、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。

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.