Development of a Highly Sensitive Cell-Based Assay for Botulinum Neurotoxin

描述（由申请人提供）：肉毒杆菌神经毒素（BoNT）是最有效的天然毒素，是一种潜在的食品污染物，也是一种宝贵的医疗药物。尽管BoNT/A和BoNT/B在临床应用中得到了有效的利用，但其主要的不良反应是产生抗体，使患者难以治疗。目前，患者在治疗期间没有常规监测抗体的形成，因为用于测量人血清（以及其他动物物种，如马）抗体的灵敏测定法尚未上市。小鼠生物测定相对不敏感，并且有众所周知的缺点，包括需要动物和相关的必要设施，结果需要2-4天，非特异性死亡，以及下面更详细描述的其他缺点。包括ELISA和体内催化活性测定在内的其他检测平台都有高背景的缺点，重要的是它们只测量BoNT活性的一种生物学特性。本研究项目的总体假设是，通过对细胞系的特异性评估，以及对细胞系中BoNT毒性重要的细胞因子的过度表达，可以开发出一种基于细胞的检测方法，用于敏感和定量地检测BoNT和BoNT的血清抗体或抑制剂。本提案的目标是设计一种对BoNT/ a高度敏感的连续细胞系，并可以测量BoNT对神经细胞中毒的各个方面。该细胞系将为小鼠生物测定提供一种特异性、敏感性、定量、相对快速和低成本的替代方法。除了用于筛选人血清中是否存在BoNT/A抗体外，这种基于细胞的灵敏BoNT检测方法还有许多其他潜在的应用，包括大规模筛选新的BoNT抑制剂，评估BoNT/A毒素的效力，以及作为一种研究工具。目前可用的针对bont的抗毒素很少，而且严重副作用的发生率很高。因此，迫切需要鉴定和生产新的、更安全的BoNT抗毒素和抑制剂。为了构建这样的细胞系，我们将研究BoNT/ a细胞表面受体（突触囊泡蛋白2）在神经2a细胞和PC12细胞等细胞系中过表达的影响。一种或两种有希望的细胞系将通过与BoNT敏感性相关的几种因子的短暂细胞内表达来评估其增强敏感性的能力，这些因子包括SV2c、gd3合成酶（诱导神经节苷脂的形成）和synaptotagmin I/II。将优化检测条件以提高灵敏度。优化将包括测试细胞内荧光BoNT传感器的灵敏度，并可能通过稳定表达这些传感器来增强细胞系。最后，将细胞实验与BoNT小鼠实验进行比较。公共卫生相关性：本提案的目标是构建对生物活性BoNT/ a高度敏感的细胞系。该细胞系可用于大规模筛选项目，旨在发现BoNT抑制剂，以及在BoNT测试中的许多其他潜在应用。虽然该试验不会完全取代体内小鼠生物试验，但作为筛选项目和其他涉及BoNT/ a的研究的第一步，它将非常有价值，因此将大大减少用于BoNT试验的动物数量。这种细胞系在检测经bont治疗的患者的中和抗体方面也可能被证明是非常有用的，这是bont医学应用中一个持续存在的问题。

DESCRIPTION (provided by applicant): Botulinum neurotoxin (BoNT), the most potent naturally occurring toxin, is a potential food contaminant but also an invaluable medical drug. Despite their effective use of BoNT/A and BoNT/B in clinical applications, the major adverse effect has been the formation of antibodies, which make patients refractory to treatment. Currently, patients are not routinely monitored for antibody formation during their treatment regime since a sensitive assay that measures antibodies in human sera (and in other animal species such as equine) is not commercially available. The mouse bioassay is relatively insensitive and has well-known drawbacks including the need for animals and associated required facilities, the requirement for 2-4 days for results, nonspecific deaths, and other drawbacks as described in more detail below. Other detection platforms including ELISA and determination of catalytic activity in vivo have the drawback of high background and importantly that they measure only one biological property of BoNT activity. The overall hypothesis of this research project is that a cell-based assay can be developed for sensitive and quantitative detection of BoNTs and serum antibodies or inhibitors to BoNTs by the specific evaluation of cell lines, and by over-expression of cell factors important for BoNT toxicity in the cell line. The goal of this proposal is to design a continuous cell line that is highly sensitive to BoNT/A and which would measure all aspects of neuronal cell intoxication by BoNT. This cells line would provide a specific, sensitive, quantitative, relatively fast and low-cost alternative to the mouse bioassay. In addition to its use in screening of human serum for the presence of antibodies to BoNT/A, such a sensitive cell-based assay for BoNT will have many other potential applications, including large-scale screenings for new BoNT inhibitors, evaluating BoNT/A toxin potency, as well as serving as a research tool. Currently available antitoxins against BoNTs are scarce and have a high incidence of serious side effects. Therefore, there is a great need for the identification and production of new and safer BoNT antitoxins and inhibitors. In order to construct such a cell line, the effect of over-expression of the BoNT/A cell surface receptor (synaptic vesicle protein 2) in cell lines such as neuro-2a cells and PC12 cells will be examined. One or two promising cell lines will be evaluated for the ability to enhance sensitivity by transient intracellular expression of several factors implicated in BoNT sensitivity, including SV2c, gD3-synthase (which induces formation of gangliosides), and synaptotagmin I/II. Assay conditions will be optimized to increase sensitivity. Optimization will include testing the sensitivity of fluorescent BoNT sensors inside the cells and possibly enhancing the cell line by stably expressing these sensors. Finally, the cell assay will be compared to the BoNT mouse assay. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to construct a cell line that is highly sensitive to biologically active BoNT/A. This cell line could then be used in large-scale screening projects designed to discover BoNT inhibitors, as well as numerous other potential applications in BoNT testing. While this assay will not entirely replace the in vivo mouse bioassay, it would be very valuable as a first step in screening projects and other research involving BoNT/A, and therefore would significantly reduce the number of animals used in BoNT assays. This cell line might also prove extremely useful in the detection of neutralizing antibodies in BoNT-treated patients, which is an ongoing problem with the medical use of BoNTs.