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

描述（由申请方提供）：为了满足FDA对制造产品中肉毒杆菌神经元毒素A（BoNT/A）进行可靠评估的要求，我们建议应用荧光肽信标技术，在单次测定中测量毒素的结合功能和蛋白水解功能，因此“从实验室到市场”。因为这是一种同质技术，所以它具有易用性、便携性、自动化和高通量。在这里，我们提出开发一系列新型荧光试剂，将它们与固定化抗体结合在自含系统中，以实现复杂样品中BoNT/A的稳健和高灵敏度检测。具体而言，我们的目标如下：具体目标#1：设计、合成和测试作为示踪剂的新型荧光肽信标，以检测毒素的结合功能;具体目标#2：设计、合成和测试新型荧光肽，以检测毒素的蛋白水解功能;具体目标#3：设计、合成和测试由上述两种肽通过柔性接头连接在一起制成的新型融合肽，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：修改现有便携式荧光读数器/培养箱，用作测量荧光动力学的试验台，并将此类测量与效价单位相关联。根据我们在这类系统上的工作经验，我们相信所提出的概念将被证明是可行的。我们希望拟议的工作将奠定一个良好的基础，启动该项目的下一个发展阶段，在那里我们将建立体外测试结果与小鼠生物测定使用复杂的样品基质和开发强大的仪器的相关性。我们的长期目标是寻求FDA批准来制造和销售这种方法和设备，用于在制造工厂、监管机构、世界卫生组织、医院和医生办公室、边境控制等中的常规部署。 公共卫生相关性：肉毒梭菌神经毒素（BoNTs）是一类广泛存在于自然界的肉毒梭菌（Clostridium botulinum）产生的神经毒素。虽然这些毒素在意外感染、食物中毒或生物攻击事件中可能导致疾病和伤亡，但它们也被广泛用于治疗疾病，改善75个以上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，以准确测量制造产品中毒素的效力，以帮助防止剂量过高或过低。当完全开发后，该系统可以部署在制造厂，监管机构，医院和医生办公室，世界卫生组织，边境管制等。

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.