Robust Peptide-Based Diagnostics of Botulinum Toxins

基于肽的肉毒杆菌毒素的稳健诊断

基本信息

批准号：
8432962
负责人：
JOHN E MUELLER
金额：
$ 25万
依托单位：
LYNNTECH, INC.
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-24 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8432962
关键词：
Abrin Adult Affinity Air Amino Acids Anaerobic Bacteria Antibodies Bacteria Bacterial Toxins Benchmarking Binding Proteins Bioinformatics Biological Biological Assay Biological Markers Biological Monitoring Bioterrorism Blood Circulation Bontoxilysin Botulinum Toxin Type A Botulinum Toxins Categories Cells Centers for Disease Control and Prevention (U.S.)Clinical Clinical Treatment Clostridium botulinum Clostridium enterotoxin Coagulation Process Custom Detection Development Devices Diagnosis Diagnostic Disease Dose Dreams Early Diagnosis Elements Environmental Pollution Enzyme-Linked Immunosorbent Assay Enzymes Epidemic Equipment Escherichia coli Event Exposure to Fluorescence Food Supply Future Generations Genus staphylococcus Goals Health Health Benefit Human Human Resources Immunoassay In Situ In Vitro Industrial Accidents Institutes Intoxication Label Laboratory Diagnosis Lethal Dose 50 Ligands Link Measures Medicine Methods Monitor Mus Muscle National Institute of Allergy and Infectious Disease Neurotoxins Online Systems Organism Paralysed Patients Peptides Phage Display Phase Preventive Production Proteins Public Health Reader Reagent Refrigeration Relative (related person)Ricin Running Serotyping Shiga-Like Toxins Silicon Technology Therapeutic Time Toxic effect Toxin Toxoids Training Transistors Water aerosolized base biothreat botulinum cancer cell cost density design improved in vivo luminescence manufacturing scale-up microbial milligram multiplex detection nanowire novel pathogen point of care point-of-care diagnostics prophylactic rapid diagnosis sensor

项目摘要

DESCRIPTION (provided by applicant): The botulinum neurotoxins (BoNTs; A-G), secreted by the anaerobic bacterium Clostridium botulinum, are highly lethal microbial proteins with an extremely low half-lethal dose of only 1-3 ng/kg in humans. In other words, one hundredth of a milligram (10 ¿g) of the toxin could be extremely fatal to an adult; lower doses can result in partial muscle paralysis. Of the seven serotypes (A-G), only types A, B, E, and F are known to be pathogenic in humans. Owing to their high toxicity and to the ease of their production and potential dissemination into air, water, and food supply, the BoNTs are considered among the preeminent bioterrorism threats. As such, the NIAID and CDC have categorized them as Class A threat agents. The benchmark BoNT assay is based on an in vivo mouse lethality assay which can detect the BoNTs at very low levels (10 pg/mL). However, besides being viewed as inhumane and costly to run, the assay results are not available for 2-4 days. This is clearly untenable in a potential bioterrorism situation because, in order to implement suitable therapeutic and prophylactic measures in the event of an intentional release, it is critical that rapid and early diagnosis of neurotoxin intoxication in humans be possible. More pertinently, the assay is more suited for detecting environmental contamination of the toxin than for human biomonitoring. Routine laboratory diagnosis of botulinum intoxication is based on the detection of the neurotoxin in the patient. In vitro diagnostic immunoassays, such as ELISA, enzyme-linked coagulation assay, and IPCR (immunoPCR) methods have been developed for this. However, nearly all such methods are confounded by one or the other of the following requirements: 1) expensive and/or sensitive reagents (antibodies); that require stringent storage (e.g., refrigeration) and delicate assay conditions; 2) protracted assay time; 3) limited sensitiviy of detection; 4) bulky detection equipment (e.g., fluorescence or luminescence plate reader); and/or 5) trained personnel to execute assays. Assays that monitor functional proteolytic activity of the neurotoxins have also been developed, many of which are limited by similar constraints. Consequently, the utility of these for point-of-care diagnosis of BoNT intoxication in humans is limited. This Phase I proposal describes the development of highly robust, short peptide molecules with exquisite affinity for the BoNTs that can serve as antibody replacements in field- deployable diagnostics. The novel discovery approach will find broad impact for designing short peptide affinity reagents to multiple other proteinaceous biothreat agents, including shiga and shiga-like toxins, Staphylococcus and Clostridium enterotoxins, abrin, and ricin. Long term, we envisage the incorporation of short, high-affinity peptides into a highly-sensitive, label-free, multiplexed electrical detection platform, premised on silicon nanowire field-effect transistors, suitable for the generation of low cost, low power, easy-to- use handheld devices for rapid monitoring and detection of toxins produced by pathogenic organisms in humans. PUBLIC HEALTH RELEVANCE: The availability of field-deployable, multiplexed diagnostic devices capable of rapidly detecting multiple critical biological threat agents, such as bacterial toxins, in the field is premised on the availability of low cost, robust, and stable capture reagens integrated with a portable but ultrasensitive detection platform. Such a capability will enable prompt treatment and/or preventative strategies to be instituted expeditiously for maximum public health benefit. In addition to toxin monitoring, our proposed multiplexed point-of-care diagnostic platform has valuable ramifications for public health by enabling truly individualized medicine.

描述（由申请人证明）：肉毒神经毒素（BONTS; a-g），厌氧细菌的thal thal微生物蛋白，其半致死剂量仅为1-3 ng/kg人类，换句话说，一百毫克。（10 g）毒素政变对七个血清型（A -G）是极端致命的，只有A，B，E和F是人类的致病性。生物恐怖主义的威胁。在2-4天内不可用的结果，这显然是在人类中进行的肠道释放的情况下，这显然是不受欢迎的治疗方法。肉毒杆菌的硼酸诊断是基于对患者的神经毒素的检测。昂贵的试剂（例如，抗体）；因此，要执行测定发现其他蛋白质生物疗法，将志贺氏毒素，葡萄球菌和类肠毒素，ABRIN和RICIN等长期，HI GH亲和肽纳米线现场效应晶体管，适用于成本的大量，低功率F毒素由人类病原生物产生的低功率F毒素。公共卫生的相关性：可用的生物学威胁剂，例如细菌毒素，在低成本的情况下，却可以使用dockinitiusitiusitius。，我们提出的通过真正个性化的医学来实现了公共卫生的促进多个可兑现的影响。