Fast accurate ultrasensitive in vitro assay to determine BoNT potency & stability

快速准确的超灵敏体外测定法测定 BoNT 效力

• 批准号: 8253009
• 负责人: Taiho Kim
• 金额: $ 11.95万
• 依托单位: NESHER TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2013-09-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8253009
• 关键词: Acetylcholine; Affinity; Animals; Antibodies; Area; Automation; Beverages; Binding; Biological Assay; Biophysics; Body Fluids; Bontoxilysin; Botulinum Toxin Type A; Botulinum Toxins; Categories; Cells; Cessation of life; Cleaved cell; Clinical; Color; Communicable Diseases; Complement; Complex; Computer software; DNA; Data; Detection; Development; Diagnostic; Discrimination; Disease Outbreaks; Dose; Dyes; Endopeptidases; Energy Transfer; Enzyme-Linked Immunosorbent Assay; Event; Exclusion; FDA approved; Fluorescence; Fluorescence Spectroscopy; Food; Funding; Future; Goals; Head; Health Care Costs; Hereditary Disease; Hour; Human; In Vitro; Individual; Intellectual Property; Intraperitoneal Injections; Label; Lasers; Leadership; Lethal Dose 50; Licensing; Manufacturer Name; Measures; Metalloproteases; Methodology; Methods; Microfluidics; Miniaturization; Monitor; Mus; Noise; Nucleic Acids; Patients; Peptide Hydrolases; Peptide Synthesis; Peptides; Phase; Poison; Population; Procedures; Process; Proteins; RNA; Reader; Reading; Reagent; Reference Standards; Reproducibility; Sampling; Screening for cancer; Sensitivity and Specificity; Signal Transduction; Site; Small Business Innovation Research Grant; Societies; Solutions; Sorting - Cell Movement; Source; Spectrum Analysis; Staging; Standardization; Synaptic Vesicles; Syndrome; System; Technology; Testing; Therapeutic; Time; Toxin; Translating; Ursidae Family; Validation; Work; Zinc; base; biothreat; cost; cost effective; fluorophore; high throughput screening; improved; in vitro Assay; innovation; instrument; interest; nanobiotechnology; nanolitre; operation; potency testing; prototype; receptor binding; response; single molecule; tool; uptake; user-friendly; virtual

DESCRIPTION (provided by applicant): Botulinum Neurotoxins (BoNTs) are the most toxic substances known and classified as Category A biothreat agents. BoNTs are zinc metalloproteases that cleave and inactivate proteins involved in synaptic vesicle fusion and acetylcholine release. Therapeutic applications of botulinum toxins have increased steadily over the years requiring new and reliable assays to assess the potency and stability of BoNT products. Nesher Technologies, Inc. (NTI) has exclusively licensed the intellectual property for a revolutionary quantitative, ultrasensitive and -specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with exquisite single-well multiplexing potential, minimal sample requirements, and extremely simplified handling procedures (no separation/washing steps). It is based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, whereby two (or more) target recognition molecules are tagged with different color fluorescence dyes. Coincident confocal detection of two (or more) colors constitutes a positive target detection event, allowing identification of biomolecules in solution and detection of numerous targets simultaneously. NTI recently achieved expansion from 2-color (2c) to 4-color (4c) ALEX, substantially expanding its multiplexing power. Furthermore, recent work by our consultants Profs. Stephen Quake and Shimon Weiss demonstrates successful merging of microfluidics-based sample handling with 2c-ALEX spectroscopy for quantification of enzymatic activity, a new breakthrough approach for assay miniaturization and increased throughput termed "single molecule optofluidics". NTI's long-term goal is to develop rapid, highly multiplexed (with a capacity of >100 analytes per sample), ultrasensitive and -specific, quantitative, cost-effective, and fully automated, protein- and nucleic acid-based syndrome-driven tests that require minimal patient samples. In this Phase I SBIR application, we propose to adapt ALEX-based single molecule optofluidics for development of an innovative, completely solution-based in vitro endopeptidase assay, overcoming limitations of animal- and cell-based assays, to determine BoNT/A potency and stability based on quantifying its protease activity by measuring fluorophore-labeled substrate cleavage. Specific Aims are: 1. Substrate peptide synthesis and fluorophore/quencher conjugation. 2. BoNT/A detection and quantification using ALEX-based single molecule optofluidics. 3. Determination of specificity, sensitivity (d100 fM), linear range of quantification (e4 log orders), & time (1 hr). Phase II will be dedicated to assay optimization, automation, prototype development, and, if considered desirable, expansion to include other BoNT substrates for rapid multiplexed (single-well) discrimination and quantification. Software will be developed suitable for user-friendly operation of a fully integrated ALEX-based testing system for routine commercial release testing of BoNT products. It may serve as basis for future development of an FDA-approved diagnostic system for BoNT and other infectious diseases agents testing. PUBLIC HEALTH RELEVANCE: Nesher Technologies, Inc. intends to develop an innovative alternative approach, based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, to the currently used mouse intraperitoneal injection assay which measures activity in Mouse Units (MU), with 1 MU of activity defined as the LD50 dose (death within 72 to 96 hours in 50% of the mice), in order to allow for standardization of potency units for Botulinum Neurotoxin products made by different manufacturers. This will greatly reduce use of animals and will also complement Nesher Technologies' federally-funded efforts of instrument and reagent development for tests for bioterror agents, infectious and genetic diseases, and early cancer detection, thereby translating cutting-edge innovations in nanobiotechnology into benefits for the society at large by saving human lives, monitoring the population for bioterror attacks and disease outbreaks, and reducing healthcare costs.

描述（由申请人提供）：肉毒神经毒素（BoNT）是已知毒性最大的物质，被归类为A类生物威胁剂。BoNTs是锌金属蛋白酶，其切割和修饰参与突触囊泡融合和乙酰胆碱释放的蛋白。肉毒杆菌毒素的治疗应用多年来稳步增加，需要新的和可靠的测定来评估BoNT产品的效力和稳定性。 Nesher Technologies，Inc. (NTI)独家授权了一项革命性的定量、超灵敏和特异性生物检测技术的知识产权，该技术是在加州大学洛杉矶分校单分子生物物理实验室（由Shimon韦斯教授领导）开发的，具有精致的单孔多路复用潜力，最小的样品要求和极其简化的处理程序（无分离/洗涤步骤）。它是基于交替激光激发（ALEX）单分子荧光光谱，其中两个（或更多个）目标识别分子标记不同颜色的荧光染料。两种（或更多种）颜色的同时共聚焦检测构成阳性靶检测事件，允许同时鉴定溶液中的生物分子和检测许多靶。NTI最近实现了从2色（2c）到4色（4c）ALEX的扩展，大大扩展了其复用能力。此外，我们的顾问教授最近的工作。Stephen Quake和Shimon韦斯展示了基于微流体的样品处理与2c-ALEX光谱法的成功融合，用于定量酶活性，这是一种用于测定小型化和增加通量的新突破性方法，称为“单分子光流体”。 NTI的长期目标是开发快速、高度多路复用（每个样本的分析能力>100个）、超灵敏和特异性、定量、成本效益和全自动的、基于蛋白质和核酸的综合征驱动的测试，这些测试需要最少的患者样本。在这个I期SBIR应用中，我们建议调整基于ALEX的单分子光流体技术，以开发一种创新的、完全基于溶液的体外内肽酶测定法，克服基于动物和细胞的测定法的局限性，通过测量荧光团标记的底物裂解来定量其蛋白酶活性，从而确定BoNT/A的效价和稳定性。具体目标是：1。底物肽合成和荧光团/猝灭剂缀合。2.使用基于ALEX的单分子光流体学进行BoNT/A检测和定量。3.确定特异性、灵敏度（d100 fM）、定量的线性范围（e4对数级）和时间（1小时）。 第二阶段将致力于分析优化，自动化，原型开发，以及，如果认为需要的话，扩展到包括其他BoNT底物，用于快速多重（单孔）鉴别和定量。将开发适用于完全集成的基于ALEX的测试系统的用户友好操作的软件，用于BoNT产品的常规商业发布测试。它可以作为未来开发FDA批准的BoNT和其他传染病病原体检测诊断系统的基础。 公共卫生相关性：Nesher Technologies，Inc.旨在开发一种创新的替代方法，基于交替激光激发（ALEX）单分子荧光光谱法，以目前使用的小鼠腹膜内注射测定法（以小鼠单位（MU）测量活性，1 MU活性定义为LD 50剂量）（50%的小鼠在72至96小时内死亡），以允许由不同制造商制造的肉毒神经毒素产品的效力单位的标准化。 这将大大减少动物的使用，也将补充Nesher Technologies联邦资助的仪器和试剂开发工作，用于生物恐怖剂，传染病和遗传病以及早期癌症检测的测试，从而将纳米生物技术的尖端创新转化为整个社会的利益，拯救人类生命，监测生物恐怖袭击和疾病爆发的人口，并降低医疗保健成本。