Diagnostic assay systems for Botulinum Neurotoxins

描述(由申请人提供)：该项目的目标是生成强大的、便携式的检测系统，用于快速检测临床样本中的肉毒杆菌神经毒素(BONT)。我们的目标是在45分钟的测试时间内检测到活性的BONT，其检测极限至少比当前的金标准-活体小鼠生物检测好10倍。BONT是已知的最有效的生物毒素。当注射、吸入或摄取微量的BONT时，会在人类和其他哺乳动物中引起松弛麻痹。然而，BONT也被广泛获得并用作美容和治疗目的的医用药物。BONT产品的可获得性和潜在的滥用对公众健康构成了真正的威胁，并与我们的国家安全密切相关。我们的方法是基于具有大免疫吸附表面积(Alissa)的珠基原型试验，该方法可以检测复杂生物基质(如血清和液体食品)中A型BONT的全摩尔浓度。Bont Alissa至少比活的小鼠生物检测灵敏四个数量级，而且速度快得多，只需要几个小时，而不是几天。它也更经济，而且不需要使用实验室动物。为了建立概念验证和改进BoNT Alissa，我们提出了以下具体目标：1.研制用于BoNT A型和B型(包括亚型)微柱捕获的亲和试剂。2.设计新的底物，用于BONT触发的具有生物发光读出的两步酶级联，用于Alissa。4.在生物防御实验室反应网络的国家实验室建立A、B、C、E和F型的BONT Alissa系统，用于收集婴儿肉毒杆菌中毒的临床前数据。该项目的长期目标是以调查设备豁免的形式获得使用微柱Bont Alissa作为诊断医疗设备的监管批准。本文提出的抗BONT亲和试剂将以单链片段变量或抗原结合片段的形式产生重组抗体，并安装在微亲和柱上，我们将其放置在一次性塑料吸管的顶端。这些柱将能够免疫捕获复杂基质中的BoNTs，具有高亲和力和特异性。生物发光底物将被生产为基因工程的非活性版本的荧光素酶，这些荧光素酶由微柱基Alissa中的BoNTs激活。这项研究的产品将是一种易于使用的生化试剂盒和亲和吸管尖端，可以快速、灵敏地检测BONT，而不需要特殊的实验室设备。作为诊断工具，这种试剂盒应该可以改善公众健康，增强我们的国家生物防御准备。

DESCRIPTION (provided by applicant): The goal of this project is to generate robust, portable assay systems for the rapid detection of botulinum neurotoxin (BoNT) in clinical samples. Our objective is to detect active BoNT within a 45 min test time at a limit of detection that is at least 10-fold better than the current gold standard, the live mouse bioassay. BoNT is the most potent biological toxin known. When injected, inhaled or ingested in minute amounts, BoNT elicits flaccid paralysis in humans and other mammals. However, BoNT is also widely available and used as a medical drug for cosmetic and therapeutic purposes. The availability and potential abuse of BoNT products presents a real threat to public health, and is of great concern to our national security. Our approach is based on a bead-based prototype assay with a large immuno-sorbent surface area (ALISSA) that can detect attomolar concentrations of BoNT serotype A in complex biological matrices such as blood serum and liquid foods. The BoNT ALISSA is at least four orders of magnitude more sensitive than the live mouse bioassay and considerably faster, requiring only hours instead of days. It is also much more economical and doesn't require the use of laboratory animals. To establish proof-of concept and improve the BoNT ALISSA, we propose the following Specific Aims: 1. To generate affinity reagents for microcolumn-capture of BoNT serotypes A and B (including subtypes). 2. To engineer novel substrates for BoNT-triggered dual-step enzyme cascades with bioluminescent readout for use in the ALISSA. 3. To generate affinity reagents for BoNT ALISSA detection of serotypes C, E, and F. 4. To establish the BoNT ALISSA system for serotypes A, B, C, E and F at a state laboratory of the Biodefense Laboratory Response Network for preclinical data collection on samples of infant botulism. The long term goal of the project is to obtain regulatory approval for use of the microcolumn BoNT ALISSA as a diagnostic medical device in form of an Investigational Device Exemption. The anti-BoNT affinity reagents proposed here will be generated as recombinant antibodies in form of single chain fragment variables or antigen-binding fragments and mounted onto microaffinity columns that we place into the tips of disposable plastic pipettes. The columns will be capable of immuno-capturing BoNTs in complex matrices with high affinity and specificity. Bioluminescent substrates will be produced as genetically- engineered inactive versions of luciferases that are activated by BoNTs in the microcolumn-based ALISSA. The product of this research will be an easy to use kit of biochemical reagents and affinity pipette tips that will allow rapid, sensitive BoNT detection without the need for special laboratory equipment. Used as diagnostic tools, such kits should improve public health and enhance our national biodefense preparedness.