Diagnostic assay systems for Botulinum Neurotoxins

肉毒杆菌神经毒素的诊断测定系统

• 批准号: 8876550
• 负责人: MARKUS KALKUM
• 金额: $ 70.64万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8876550
• 关键词: Affinity; Animals; Antibodies; Area; Biochemical; Biological; Biological Assay; Body Weight; Bontoxilysin; Botulinum Toxin Type A; Botulism; Breathing; California; Categories; Chemicals; Clinical; Complex; Cosmetics; Data Analyses; Data Collection; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic Procedure; Drug Formulations; Engineering; Enzymes; Epitope Mapping; Epitopes; Equipment; Fab Immunoglobulins; Fluorescence; Food; Genetic Engineering; Goals; Gold; Head; Health Personnel; Hour; Human; Immunosorbents; Infantile Botulism; Intoxication; Laboratories; Laboratory Animals; Life; Light; Liquid substance; Living Standards; Luciferases; Mammals; Maps; Measurement; Measures; Medical; Medical Device; Metalloproteases; Methods; Monoclonal Antibodies; Mus; National Institute of Allergy and Infectious Disease; National Security; Paralysed; Peptides; Pharmaceutical Preparations; Plastics; Preparation; Prevention program; Process; Proteins; Public Health; Readiness; Reading; Reagent; Recombinant Antibody; Recombinants; Reporting; Reproducibility; Research; Research Project Grants; Sampling; Sensitivity and Specificity; Sepharose; Serotyping; Serum; Site; Specificity; Specimen; Surface; System; Techniques; Testing; Therapeutic; Time; Toxin; Training; United States Food and Drug Administration; Zinc; base; biodefense; biothreat; botulinum toxin type C; design; diagnostic assay; holotoxins; improved; inhibitor/antagonist; novel; polyclonal antibody; pre-clinical; prototype; rapid detection; response; target SNARE proteins; tool; treatment program

DESCRIPTION (provided by applicant): The goal of this project is to generate robust, portable assay systems for the rapid detection of botulinum neurotoxin (BoNT) in clinical samples. Our objective is to detect active BoNT within a 45 min test time at a limit of detection that is at least 10-fold better than the current gold standard, the live mouse bioassay. BoNT is the most potent biological toxin known. When injected, inhaled or ingested in minute amounts, BoNT elicits flaccid paralysis in humans and other mammals. However, BoNT is also widely available and used as a medical drug for cosmetic and therapeutic purposes. The availability and potential abuse of BoNT products presents a real threat to public health, and is of great concern to our national security. Our approach is based on a bead-based prototype assay with a large immuno-sorbent surface area (ALISSA) that can detect attomolar concentrations of BoNT serotype A in complex biological matrices such as blood serum and liquid foods. The BoNT ALISSA is at least four orders of magnitude more sensitive than the live mouse bioassay and considerably faster, requiring only hours instead of days. It is also much more economical and doesn't require the use of laboratory animals. To establish proof-of concept and improve the BoNT ALISSA, we propose the following Specific Aims: 1. To generate affinity reagents for microcolumn-capture of BoNT serotypes A and B (including subtypes). 2. To engineer novel substrates for BoNT-triggered dual-step enzyme cascades with bioluminescent readout for use in the ALISSA. 3. To generate affinity reagents for BoNT ALISSA detection of serotypes C, E, and F. 4. To establish the BoNT ALISSA system for serotypes A, B, C, E and F at a state laboratory of the Biodefense Laboratory Response Network for preclinical data collection on samples of infant botulism. The long term goal of the project is to obtain regulatory approval for use of the microcolumn BoNT ALISSA as a diagnostic medical device in form of an Investigational Device Exemption. The anti-BoNT affinity reagents proposed here will be generated as recombinant antibodies in form of single chain fragment variables or antigen-binding fragments and mounted onto microaffinity columns that we place into the tips of disposable plastic pipettes. The columns will be capable of immuno-capturing BoNTs in complex matrices with high affinity and specificity. Bioluminescent substrates will be produced as genetically- engineered inactive versions of luciferases that are activated by BoNTs in the microcolumn-based ALISSA. The product of this research will be an easy to use kit of biochemical reagents and affinity pipette tips that will allow rapid, sensitive BoNT detection without the need for special laboratory equipment. Used as diagnostic tools, such kits should improve public health and enhance our national biodefense preparedness.

描述（由申请人提供）：该项目的目标是生成强大的便携式检测系统，用于快速检测临床样品中的肉毒杆菌神经毒素（BoNT）。我们的目标是在45分钟的测试时间内检测到活性BoNT，其检测极限至少比目前的金标准（活体小鼠生物测定法）好10倍。BoNT是已知最强效的生物毒素。当注射、吸入或摄入微量BoNT时，会引起人类和其他哺乳动物的弛缓性麻痹。然而，BoNT也被广泛用于美容和治疗目的。BoNT产品的可用性和潜在滥用对公众健康构成了真正的威胁，并对我们的国家安全产生了重大影响。我们的方法是基于一个具有大免疫吸附表面积（ALISSA）的基于珠子的原型分析，可以检测复杂生物基质（如血清和液体食物）中BoNT血清型a的原子摩尔浓度。BoNT ALISSA的灵敏度至少比活体小鼠生物测定法高4个数量级，而且速度也快得多，只需几个小时，而不是几天。它也更经济，不需要使用实验动物。为了建立概念验证和改进BoNT ALISSA，我们提出以下具体目标：制备用于BoNT血清型A和B（包括亚型）微柱捕获的亲和试剂。2. 设计用于bont触发的双步酶级联的新型底物，并具有生物发光读数，用于ALISSA。3. 制备用于C、E、f血清型BoNT ALISSA检测的亲和试剂。在生物防御实验室反应网络的国家实验室建立血清A、B、C、E和F型BoNT ALISSA系统，用于收集婴儿肉毒杆菌中毒样本的临床前数据。该项目的长期目标是获得监管部门批准，以研究设备豁免的形式使用微柱BoNT ALISSA作为诊断医疗设备。本文提出的抗bont亲和试剂将以单链片段变量或抗原结合片段的形式产生重组抗体，并安装在我们放置在一次性塑料移液管尖端的微亲和柱上。该色谱柱将能够在具有高亲和力和特异性的复杂基质中免疫捕获bont。生物发光底物将作为荧光素酶的基因工程无活性版本产生，这些荧光素酶在微柱基ALISSA中被bont激活。这项研究的产品将是一个易于使用的生化试剂试剂盒和亲和力移液头，将允许快速，灵敏的BoNT检测，而不需要特殊的实验室设备。作为诊断工具，这类试剂盒将改善公众健康，加强我们的国家生物防御准备。