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（由适用提供）：由厌氧菌细菌分泌的肉毒杆菌神经毒素（BONTS； A-G）是肉毒杆菌梭菌的分泌，是高度致命的微生物蛋白，其仅为1-3 ng/kg的肉毒杆菌蛋白质是高度致死的微生物蛋白。换句话说，毒素的一百毫克（10 g）可能对成年人致命。较低剂量会导致部分肌肉瘫痪。在七种血清型（A-G）中，只有A，B，E和F型在人类中是致病性的。由于它们的高毒性以及其生产的便利性以及潜在的空气，水和粮食供应的传播，因此在杰出的生物恐怖主义威胁中被认为是bonts。因此，NIAID和CDC将它们归类为A类威胁代理商。基准BONT测定基于体内小鼠致死性测定法，该测定可以检测到非常低的水平（10 pg/ml）的BONT。但是，除了被视为不人道和昂贵的运行外，评估结果在2-4天内都无法使用。在潜在的生物恐怖情况下，这显然是站不住脚的，因为为了实施适当的治疗和预防性措施，在有意释放的情况下，至关重要的是，可以在人类中快速和早期诊断神经毒素中毒。更重要的是，该评估更适合于检测毒素的环境污染，而不是人类的生物监测。肉毒杆菌中学的常规实验室诊断是基于患者中神经毒素的检测。为此开发了体外诊断免疫测定，例如ELISA，酶联凝血测定法和IPCR（IMPCR）方法。但是，几乎所有此类方法都被以下一个或另一种要求混淆：1）昂贵和/或敏感试剂（抗体）；这需要严格的存储（例如，制冷）和微妙的测定条件； 2）长期的测定时间； 3）有限的检测灵敏度； 4）笨重的检测设备（例如荧光或发光板读取器）；和/或5）经过训练的人员执行测定。也已经开发了监测神经毒素功能有水解活性的测定法，其中许多受到相似约束的限制。因此，这些实用性用于人类BONT肠毒性诊断的诊断。这一阶段I提出了具有高度稳健的短肽分子的发展，具有对可以作为现场可部署诊断的抗体替代物的独家亲和力的独家亲和力。这种新颖的发现方法将发现对在其他多种其他蛋白质生物治疗剂中设计短肽亲和力试剂，包括志贺和志公式的毒素，葡萄球菌和肠肠毒素，ABRIN和RICIN。长期，我们设想将短，高亲和力的肽掺入高度敏感的，无标签的，多重的电气检测平台中，前提是在硅纳米线现场效应晶体管上的前提，适用于生成低成本，低功率，易于使用的手持设备，可快速监测和检测由毒素产生的毒素，这些疾病是由人为生成的人类构成的。 公共卫生相关性：可在现场迅速检测到多种关键生物学威胁剂（例如细菌毒素）的可用场，多重诊断设备的可用性，以低成本，健壮和稳定的捕获试剂的可用性为前提。这样的能力将迅速治疗和/或预防策略迅速制定，以获得最大的公共卫生益处。除了毒素监测外，我们提出的多重诊断平台通过实现真正的个性化医学来对公共卫生产生宝贵的影响。