Fluorescent and bioluminescent assays for botulinum neurotoxin

这项建议的目标是开发新的生化方法和设备来检测大脑皮层肿瘤 肉毒杆菌神经毒素(BONT)的所有七个血清型和亚型。根据我们的初步数据，我们 假设可以同时产生抗药性的荧光和生物发光基质 非BONT蛋白水解性切割，但可被BoNTs快速激活。建议的具体目标 研究内容如下：1)开发A-G型BONT的新型荧光底物。 将通过具有蛋白生成和非蛋白生成氨基酸的多肽库进行设计，并将 选择用于抵抗非BONT蛋白水解酶和高效切割BoNTs。我们将测试ALISSA 用这些新底物在血清、固体器官提取物、灌肠和大便的各种骨钙素添加的样品中 标本以及各种食物。2)肉毒杆菌发光蛋白底物的研制 毒素。我们正在提议对重组荧光素酶蛋白的变种进行基因工程，这些变种可以成为 由肉毒杆菌毒素的金属蛋白酶活性介导的特定切割反应激活。 将为所有七种血清型(A到G)的肉毒杆菌毒素创建发光底物。我们还将 生物发光分析在多个波长发射光，用于多路同时检测更多 而不是每个样本有一种血清型。3)现有的和新型的BONT ALISSA在毒素检测中的应用 在动物血清和器官中的分布。与美国国务院的路易莎·郑博士合作 在加利福尼亚州奥尔巴尼的农业，我们将利用Bont Alissa来测量系统内容物和组织 亚致死性骨中毒小鼠体内的分布。我们的目标是确定药物动力学和血清型依赖 肠外或口服中毒后毒素在器官中的分布。 此外，与Intrative BioProbe的Nedelkov博士和Precision的Chris Myatt博士合作 我们将开发和测试全自动设备，利用我们的新方法 全血过敏性BNTs的定量检测。邦特·艾丽莎的技术将被改造成 形成整体式微柱形式，以供普通液体处理机器人使用。由此产生的台式机 仪器还将采用高灵敏度和廉价的基于波导型的光学读取器 系统。我们的检测系统将用复杂的生物基质进行测试和验证，如血清， 固体器官和食物提取物以及临床样本(在加利福尼亚州斯蒂芬·阿农博士的实验室 公共卫生署)。

The goal of this proposal is to develop novel biochemical methods and devices for the attomolar detection of all seven serotypes and subtypes of botulinum neurotoxin (BoNT). Based on our preliminary data, we hypothesize that it is feasible to generate both fluorescent and bioluminescent substrates that are resistant to non-BoNT proteolytic cleavage, but can rapidly be activated by BoNTs. The specific aims of the proposed research are: 1) Development of novel fluoroaenic substrates for BoNT serotypes A to G. These substrates will be designed through peptide libraries with proteinogenic and non-proteinogenic amino acids and will be selected for resistance to non-BoNT proteases and highly efficient cleavage by BoNTs. We will test ALISSAs with those novel substrates in various BoNT-spiked samples of serum, solid organ extracts, enema and stool specimens as well as a variety of foods. 2) Development of a luminogenic protein substrate for botulinum toxin. We are proposing to genetically engineer variants of recombinant luciferase proteins that become activated by specific cleavage reactions mediated by the metalloprotease activity of botulinum toxin. Luminescent substrates will be created for all seven serotypes (A to G) of botulinum toxin. We will also bioluminescent assays that emit light at multiple wavelenght for multiplexed simultaneous detection of more than one serotype per sample. 3) Application of existing and novel BoNT ALISSAs to measure toxin distribution in animal sera and organs. In collaboration with Dr. Luisa Cheng at the U.S. Department for Agriculture in Albany, CA, we will utilize BoNT ALISSA to measure the systemic content and tissue distribution in sub-lethally BoNT-intoxicated mice. Our goal is to determine pharmacokinetics and serotypedependent toxin distributions in organs following parenteral or oral intoxication. Furthermore, in collaboration with Dr. Nedelkov at Intrinsic Bioprobes and with Dr. Chris Myatt at Precision Photonics we will develop and test fully automated devices that will utilize our novel methodology for the quantitative detection of BoNTs at attomolar sensitivity. The BoNT ALISSA technology will be adapted into into a monolithic microcolumn format to be utilized by common liquid handling robots. The resulting benchtop instrumentation will also incorporate a highly sensitive, and inexpensive wave-guide-based optical readout system. Our assay system will be tested and validated with complex biological matrices such as serum, solid organ and food extracts as well as clinical samples (at Dr. Stephen Arnon's laboratory at the California Department for Public Health).