Robust Peptide-Based Diagnostics of Botulinum Toxins

描述（由申请人提供）：肉毒杆菌神经毒素（BoNT; A-G）由厌氧细菌肉毒梭菌分泌，是高度致死的微生物蛋白，在人体中的半致死剂量极低，仅为1-3 ng/kg。换句话说，百分之一毫克（10 μ g）的毒素对成年人来说可能是致命的;较低的剂量可能导致部分肌肉麻痹。在七种血清型（A-G）中，已知仅A、B、E和F型对人类具有致病性。由于其高毒性和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将其归类为A类威胁因子。基准BoNT测定基于体内小鼠致死性测定，其可以检测非常低水平（10 pg/mL）的BoNT。然而，除了被视为不人道和昂贵的运行，分析结果是不可用的2-4天。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，必须能够迅速和早期诊断人体神经毒素中毒。更相关的是，该测定法更适合于检测毒素的环境污染，而不是人类生物监测。肉毒杆菌中毒的常规实验室诊断是基于对患者神经毒素的检测。已经为此开发了体外诊断免疫测定，例如ELISA、酶联凝固测定和IPCR（免疫PCR）方法。然而，几乎所有这些方法都受到以下要求中的一个或另一个的干扰：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）延长的测定时间; 3）有限的检测灵敏度; 4）庞大的检测设备（例如，荧光或发光板读数器）;和/或5）受过训练的人员以执行测定。还开发了监测神经毒素的功能性蛋白水解活性的测定法，其中许多测定法受到类似约束的限制。因此，这些用于人类BoNT中毒的即时诊断的效用是有限的。该I期提案描述了对BoNT具有精确亲和力的高度稳健的短肽分子的开发，所述短肽分子可以在现场可部署的诊断中充当抗体替代物。这种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，包括滋贺和志贺样毒素、葡萄球菌和梭菌肠毒素、相思豆毒素和蓖麻毒素。从长远来看，我们设想将短的、高亲和力的肽掺入到高灵敏度的、无标记的、多路复用的电检测平台中，该电检测平台安装在硅纳米线场效应晶体管上，适合于产生低成本、低功率、易于使用的手持设备，用于快速监测和检测由人类中的病原生物体产生的毒素。 公共卫生相关性：能够在现场快速检测多种关键生物威胁剂（例如细菌毒素）的可现场部署的多路复用诊断装置的可用性取决于与便携式但超灵敏的检测平台集成的低成本、稳健且稳定的捕获试剂的可用性。这种能力将使迅速采取及时治疗和/或预防战略，以最大限度地造福公众健康。除了毒素监测，我们提出的多路复用床旁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。

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.