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.

描述（申请人提供）：肉毒杆菌神经毒素（BoNT；A-G）由厌氧菌肉毒杆菌分泌，是一种高度致死的微生物蛋白，对人体的半致死剂量极低，仅为 1-3 ng/kg。换句话说，百分之一毫克（10 µg）的毒素对成年人来说可能是极其致命的；较低剂量可导致部分肌肉麻痹。在七种血清型 (A-G) 中，只有 A、B、E 和 F 型已知对人类致病。由于其高毒性、易于生产以及可能传播到空气、水和食品供应中，BoNT 被认为是最突出的生物恐怖主义威胁之一。因此，NIAID 和 CDC 将它们归类为 A 级威胁代理。基准 BoNT 测定基于小鼠体内致死性测定，可检测极低水平 (10 pg/mL) 的 BoNT。然而，除了被视为不人道且运行成本高昂之外，检测结果还要 2-4 天才能获得。这在潜在的生物恐怖主义情况下显然是站不住脚的，因为为了在故意释放的情况下实施适当的治疗和预防措施，快速、早期诊断人类神经毒素中毒至关重要。更相关的是，该测定更适合检测毒素的环境污染，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对患者体内神经毒素的检测。为此开发了体外诊断免疫测定法，例如 ELISA、酶联凝血测定法和 IPCR (immunoPCR) 方法。然而，几乎所有此类方法都受到以下一个或另一个要求的困扰：1）昂贵和/或敏感的试剂（抗体）；需要严格的储存（例如冷藏）和精密的测定条件； 2) 检测时间延长； 3）检测灵敏度有限； 4) 笨重的检测设备（例如荧光或发光读板机）；和/或 5) 受过培训的人员执行检测。监测神经毒素功能性蛋白水解活性的测定方法也已开发出来，其中许多方法都受到类似的限制。因此，这些方法对于人类 BoNT 中毒的即时诊断的效用受到限制。该第一阶段提案描述了对 BoNT 具有极高亲和力的高度稳健的短肽分子的开发，可作为现场部署诊断中的抗体替代品。新的发现方法将对设计针对多种其他蛋白质生物威胁剂的短肽亲和试剂产生广泛影响，包括志贺和志贺样毒素、葡萄球菌和梭状芽胞杆菌肠毒素、相思豆毒素和蓖麻毒素。从长远来看，我们设想将短的、高亲和力的肽整合到一个高灵敏度、无标记、多重电检测平台中，该平台以硅纳米线场效应晶体管为前提，适合生成低成本、低功耗、易于使用的手持设备，用于快速监测和检测人类病原生物产生的毒素。 公共卫生相关性：能够在现场快速检测多种关键生物威胁因子（例如细菌毒素）的可现场部署的多重诊断设备的可用性，前提是能够提供与便携式但超灵敏检测平台集成的低成本、强大且稳定的捕获试剂。这种能力将使能够迅速制定及时治疗和/或预防策略，以实现最大的公共健康利益。除了毒素监测之外，我们提出的多重即时诊断平台通过实现真正的个体化医疗，对公共卫生产生了宝贵的影响。