Robust Peptide-Based Diagnostics of Botulinum Toxins

基于肽的肉毒杆菌毒素的稳健诊断

• 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
• 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

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.

说明(申请人提供)：肉毒杆菌神经毒素(BoNTs；A-G)由厌氧菌肉毒杆菌分泌，是一种高度致命的微生物蛋白，对人类的半数致死量极低，仅为1-3 ng/kg。换句话说，百分之一毫克(10g)的毒素对成年人来说可能是极其致命的；较低的剂量可能导致部分肌肉瘫痪。在七个血清型(A-G)中，只有A、B、E和F型已知在人类中是致病的。由于它们的高毒性以及它们易于生产和可能传播到空气、水和食物供应中，BoNTs被认为是最突出的生物恐怖主义威胁之一。因此，NIAID和CDC将他们归类为A级威胁代理。基准的BONT试验是基于体内小鼠致死性试验，该试验可以在非常低的水平(10pg/mL)检测到BoNTs。然而，除了被视为不人道和昂贵的运行，化验结果在2-4天内无法获得。在潜在的生物恐怖主义情况下，这显然是站不住脚的，因为为了在故意释放的情况下采取适当的治疗和预防措施，能够快速和早期地诊断人类神经毒素中毒是至关重要的。更准确地说，该检测方法更适合于检测环境中的毒素污染，而不是人体生物监测。肉毒杆菌中毒的常规实验室诊断是基于患者体内神经毒素的检测。为此，已发展了体外诊断免疫分析方法，如ELISA法、酶联凝血法和免疫聚合酶链式反应(IPCR法)。然而，几乎所有这些方法都受到以下要求的影响：1)昂贵和/或敏感的试剂(抗体)；需要严格的存储(例如，冷藏)和精细的检测条件；2)延长的检测时间；3)有限的检测灵敏度；4)笨重的检测设备(例如，荧光或发光平板阅读器)；和/或5)受过执行检测的培训的人员。监测神经毒素功能蛋白分解活性的分析也已经开发出来，其中许多受到类似限制的限制。因此，这些用于诊断人类BONT中毒的护理点的效用是有限的。这项第一阶段的提案描述了开发对BoNTs具有精致亲和力的高度健壮的短肽分子，可以作为现场可部署诊断中的抗体替代品。这一新的发现方法将对设计短肽亲和试剂对其他多种蛋白质类生物制剂产生广泛影响，包括志贺和志贺样毒素、葡萄球菌和梭状芽孢杆菌肠毒素、abrin和蓖麻毒素。从长远来看，我们设想将短、高亲和力的多肽整合到一个以硅纳米线场效应晶体管为前提的高灵敏度、无标记的多路电子检测平台中，适合于生成低成本、低功耗、易于使用的手持设备，用于快速监测和检测人体内病原体产生的毒素。 与公共卫生相关：能够在现场快速检测多种关键生物威胁因素(如细菌毒素)的可现场部署的多路诊断设备的可用性，是以低成本、坚固和稳定的捕获试剂与便携式但超灵敏的检测平台集成为前提的。这种能力将使迅速制定治疗和/或预防战略能够最大限度地惠及公众健康。除了毒素监测外，我们提议的多路复用式医疗点诊断平台通过实现真正的个性化医疗，对公共卫生具有宝贵的影响。