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

肉毒杆菌神经毒素高灵敏细胞检测方法的开发

• 批准号: 7660732
• 负责人: Eric A. Johnson
• 金额: $ 18.56万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-08 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7660732
• 关键词: Adverse effects; Animals; Antibodies; Antibody Formation; Antitoxins; Biological; Biological Assay; Bontoxilysin; Botulinum Toxin Type A; Botulinum Toxins; Buffers; Cell Line; Cell Surface Receptors; Cells; Cessation of life; Clinical Trials; Code; Detection; Development; Educational workshop; Enzyme-Linked Immunosorbent Assay; Equus caballus; Evaluation; Exhibits; Food; Gangliosides; Genes; Genome; Goals; Human; Incidence; Intoxication; Laboratories; Measurement; Measures; Medical; Methodology; Methods; Military Personnel; Modeling; Monitor; Motor Neurons; Mus; National Institute of Environmental Health Sciences; Neurons; PC12 Cells; Patients; Pharmaceutical Preparations; Population; Preparation; Production; Property; Proteins; Publications; Publishing; Rattus; Refractory; Regulation; Research; Research Personnel; Research Project Grants; SNAP receptor; Screening procedure; Serum; Spinal Cord; Staging; Stem cells; Synaptic Vesicles; System; Testing; Time; Toxic effect; Toxin; United States National Institutes of Health; Work; base; botulinum toxin type B; cell type; clinical application; continuous cell line; cost; design; immortalized cell; in vivo; inhibitor/antagonist; meetings; neutralizing antibody; public health relevance; receptor; sensor; stable cell line; synaptotagmin; synaptotagmin I; tool; trafficking

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.

描述（由申请人提供）：最有效的天然毒素肉毒杆菌神经毒素（BONT）是一种潜在的食物污染物，但也是一种宝贵的医疗药物。尽管它们在临床应用中有效地使用了BONT/A和BONT/B，但主要的不利影响是抗体的形成，这使患者难以治疗。目前，由于无法在其治疗方案中常规监测患者的抗体形成，因为敏感的测定方法可在人类血清中测量抗体（以及其他动物物种（如马）中的抗体）。小鼠生物测定相对不敏感，并且具有众所周知的缺点，包括对动物和相关所需的设施的需求，结果为2-4天的结果，非特异性死亡以及其他详细描述的其他缺点。其他检测平台在内，包括ELISA和体内催化活性的确定具有高背景的缺点，重要的是它们仅测量BONT活动的一种生物学特性。该研究项目的总体假设是，可以通过特定评估细胞系以及对细胞因子的过表达对细胞系的敏感和定量检测进行敏感和定量检测对BONT的敏感和定量检测，或者对BONT的抑制剂进行抑制作用。该建议的目的是设计一种对BONT/A高度敏感的连续细胞系，并通过BONT测量神经元细胞中毒的所有方面。该细胞系将提供特定的，敏感的，定量的，相对较快和低成本的替代品。除了用于筛选人血清的BONT/A抗体的存在之外，基于BONT的敏感细胞测定法还将具有许多其他潜在的应用，包括用于新的BONT抑制剂的大规模筛选，评估BONT/A A毒素效力，以及作为研究工具。目前，针对BONT的可用抗毒素是稀缺的，并且副作用的发生率很高。因此，非常需要鉴定和生产新的，更安全的抗毒素和抑制剂。为了构建这种细胞系，将检查BONT/A细胞表面受体（突触囊泡蛋白2）在细胞系（例如Neuro-2a细胞和PC12细胞）中过表达的影响。将评估一种或两个有希望的细胞系，以通过与BONT敏感性相关的几个因子的瞬时细胞内表达来增强灵敏度，包括SV2C，GD3-伴酶（诱导神经节苷脂的形成）和Synaptotototagagmin I/II。测定条件将被优化以提高灵敏度。优化将包括测试细胞内部荧光灯传感器的灵敏度，并通过稳定表达这些传感器来增强细胞系。最后，将细胞测定与BONT小鼠分析进行比较。公共卫生相关性：该提案的目的是构建一种对生物活性BONT/A高度敏感的细胞系。然后，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。尽管该测定法不能完全替代体内小鼠生物测定法，但作为筛选项目和其他涉及BONT/A的研究的第一步将非常有价值，因此将大大减少BONT分析中使用的动物数量。该细胞系也可能证明在检测经BONT治疗的患者中和抗体的情况下非常有用，这是BONTS医疗使用的持续问题。