Diagnostic assay systems for Botulinum Neurotoxins

肉毒杆菌神经毒素的诊断测定系统

• 批准号: 8184637
• 负责人: MARKUS KALKUM
• 金额: $ 79.11万
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8184637
• 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; 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; 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. PUBLIC HEALTH RELEVANCE: The product of this research project is a kit of reagents and devices for the detection of botulinum neurotoxins in biological specimens. It is relevant to public health as it will improve the diagnosis of botulism, by being substantially more sensitive, much more rapid, and more economical. Because botulinum neurotoxins are also conceived as biothreat agents, this research product will be installed in the laboratory response network and will thereby enhance our national biodefense preparedness.

描述（由申请人提供）：本项目的目标是生成用于快速检测临床样本中肉毒杆菌神经毒素（BoNT）的耐用便携式检测系统。我们的目标是在45分钟的测试时间内检测到活性BoNT，其检测限至少是目前金标准（活小鼠生物测定法）的10倍。BoNT是已知最强的生物毒素。当注射，吸入或摄入微量时，BoNT会在人类和其他哺乳动物中引起弛缓性麻痹。然而，BoNT也被广泛使用，并用作美容和治疗目的的医疗药物。BoNT产品的可用性和潜在的滥用对公共健康构成了真实的威胁，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基于珠的原型测定与一个大的免疫吸附表面积（ALISSA），可以检测阿摩尔浓度的BoNT血清型A在复杂的生物基质，如血清和液体食品。BoNT ALISSA的灵敏度比活小鼠生物测定至少高出四个数量级，而且速度也快得多，只需要几个小时而不是几天。它也更经济，不需要使用实验室动物。为了建立概念验证和改进BoNT ALISSA，我们提出了以下具体目标：1。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ALISSA的BoNT触发的双步骤酶级联反应的新型底物，并具有生物发光读数。3.为了生成用于BoNT ALISSA检测血清型C、E和F的亲和试剂。4.在生物防御实验室反应网络的国家实验室建立血清型A、B、C、E和F的BoNT ALISSA系统，用于婴儿肉毒杆菌中毒样本的临床前数据收集。该项目的长期目标是以试验用器械豁免的形式获得微柱BoNT ALISSA作为诊断医疗器械使用的监管批准。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作为重组抗体产生，并安装在微亲和柱上，我们将其放置在一次性塑料移液管的尖端中。该柱将能够以高亲和力和特异性免疫捕获复杂基质中的BoNT。生物发光底物将作为在基于微柱的ALISSA中被BoNT激活的基因工程化的失活形式的酶产生。这项研究的产品将是一个易于使用的生化试剂和亲和移液器吸头试剂盒，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，这些工具包应该改善公共卫生，加强我们的国家生物防御准备。 公共卫生相关性：本研究项目的产品是一套用于检测生物标本中肉毒杆菌神经毒素的试剂和设备。它与公共卫生有关，因为它将通过显著更敏感、更快速和更经济来改善肉毒杆菌中毒的诊断。由于肉毒杆菌神经毒素也被认为是生物威胁剂，这一研究产品将被安装在实验室反应网络中，从而加强我们的国家生物防御准备。