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）：肉毒杆菌神经毒素 (BoNT) 是已知毒性最强的物质，被归类为 A 类生物威胁物质。 BoNT 是锌金属蛋白酶，可裂解和灭活参与突触小泡融合和乙酰胆碱释放的蛋白质。多年来，肉毒杆菌毒素的治疗应用稳步增长，需要新的可靠的检测方法来评估 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 Weiss 教授领导）开发的，具有精湛的单井多重分析潜力、最少的样品要求以及极其简化的处理程序（无分离/洗涤步骤）。它基于交替激光激发 (ALEX) 单分子荧光光谱，其中两个（或多个）目标识别分子用不同颜色的荧光染料标记。两种（或多种）颜色的重合共焦检测构成了阳性目标检测事件，允许识别溶液中的生物分子并同时检测大量目标。 NTI 最近实现了从 2 色 (2c) ALEX 到 4 色 (4c) 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 Weiss 展示了基于微流体的样品处理与 2c-ALEX 光谱的成功结合，用于量化酶活性，这是一种新的突破性方法，用于测定小型化和增加通量，称为“单分子光流体”。 NTI 的长期目标是开发快速、高度多重（每个样品可容纳 >100 种分析物）、超灵敏、特异性、定量、经济高效、全自动、基于蛋白质和核酸的综合征驱动的测试需要最少的患者样本。在此 I 期 SBIR 应用中，我们建议采用基于 ALEX 的单分子光流控技术来开发一种创新的、完全基于溶液的体外内肽酶测定，克服基于动物和细胞的测定的局限性，以确定 BoNT/A 的效力和通过测量荧光团标记的底物裂解来量化其蛋白酶活性来确定稳定性。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 的单分子光流控进行 BoNT/A 检测和定量。 3.确定特异性、灵敏度(d100 fM)、定量线性范围(e4 log order)和时间(1小时)。 第二阶段将致力于测定优化、自动化、原型开发，以及如果认为需要的话，扩展以包括其他 BoNT 底物，以进行快速多重（单孔）区分和定量。将开发适合用户友好操作完全集成的基于 ALEX 的测试系统的软件，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测试诊断系统的基础。 公共健康相关性：Nesher Technologies, Inc. 打算开发一种基于交替激光激发 (ALEX) 单分子荧光光谱的创新替代方法，以替代目前使用的小鼠腹膜内注射测定，该测定可测量小鼠单位 (MU) 的活性，其中 1活性 MU 定义为 LD50 剂量（50% 的小鼠在 72 至 96 小时内死亡），以便实现肉毒杆菌效力单位的标准化不同制造商生产的神经毒素产品。 这将大大减少动物的使用，也将补充 Nesher Technologies 联邦政府资助的生物恐怖制剂、传染病和遗传疾病以及早期癌症检测的仪器和试剂开发工作，从而将纳米生物技术的尖端创新转化为人类的利益。通过拯救人类生命、监测人口的生物恐怖袭击和疾病爆发以及降低医疗保健成本，为整个社会带来好处。