Robust Peptide-Based Diagnostics of Botulinum Toxins

描述（由申请人提供）：肉毒杆菌神经毒素（BoNTs; A-G）由厌氧细菌肉毒梭菌分泌，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克）的毒素对一个成年人来说是极其致命的；低剂量会导致部分肌肉瘫痪。在7种血清型（A- g）中，已知只有A、B、E和F型对人类具有致病性。由于它们的高毒性和易于生产以及可能传播到空气、水和食物供应中，bont被认为是主要的生物恐怖主义威胁之一。因此，NIAID和CDC将其归类为A级威胁物质。基准BoNT检测是基于体内小鼠致死试验，可以检测到非常低水平（10 pg/mL）的BoNT。然而，除了被认为是不人道的和昂贵的运行之外，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，因为为了在发生故意释放时实施适当的治疗和预防措施，对人类神经毒素中毒进行快速和早期诊断是至关重要的。更有针对性的是，该分析更适合于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神经毒素的检测。体外诊断免疫测定，如ELISA，酶联凝血测定和IPCR（免疫pcr）方法已经为此开发。然而，几乎所有这些方法都被以下要求中的一种或另一种所混淆：1)昂贵和/或敏感的试剂（抗体）；需要严格的储存（如冷藏）和精细的分析条件；2)延长测定时间；3)检测灵敏度有限；4)笨重的检测设备（如荧光或发光读卡器）；和/或5)经过培训的人员执行化验。监测神经毒素的功能性蛋白水解活性的检测也已经开发出来，其中许多都受到类似限制。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期提案描述了高度健壮的短肽分子的开发，这些分子与bont具有良好的亲和力，可以作为可现场部署诊断的抗体替代品。这一新的发现方法将对设计短肽亲和试剂以对抗多种其他蛋白质生物威胁因子产生广泛影响，包括志贺和志贺样毒素、葡萄球菌和梭状芽胞杆菌肠毒素、蓖麻毒素和蓖麻毒素。从长远来看，我们设想将短的、高亲和力的肽结合到一个高度敏感的、无标签的、多路复用的电检测平台中，以硅纳米线场效应晶体管为前提，适用于制造低成本、低功耗、易于使用的手持设备，用于快速监测和检测人体致病菌产生的毒素。

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.