A highly sensitive replacement assay for botulinum neurotoxin type B

肉毒杆菌神经毒素（Botanicneurotoxins，BoNT）是已知的最强的毒素，其中血清型A、B、E引起人类肉毒杆菌中毒，而血清型B、C、D引起动物肉毒杆菌中毒。由梭菌属细菌产生，BoNT是高度致命的。然而，在受控条件下，一些BoNT变体（血清型）可用于许多医学应用，如神经肌肉痉挛治疗。本项目的主要目的是建立一种世界领先的、定量的体外检测BoNT B型活性的方法。这将允许替代目前用于生产与BoNT B型相关的药品的小鼠致死率试验。在先前来自NC 3R的支持下，我们基因工程改造了对BoNT/B敏感的第一个细胞系。我们还纳入了发光报告系统，允许方便检测BoNT/B活性，使用用户友好的微孔板测定。现在，使用我们的细胞系并与美国国家生物标准和控制研究所（NIBSC）合作，我们建议增加细胞系的灵敏度，并开发基于单克隆抗体的BoNT/B活性测定，其在特异性和灵敏度方面优于小鼠生物测定。将使用药物BoNT/B制剂和BoNT/B抗毒素对我们的试验进行验证。该项目的成功实施将为BoNT/B制造商提供坚定的激励，以更快，更便宜和更具体的基于细胞的方法取代小鼠LD 50测试。我们还将应用我们的技术来设计用于检测BoNT D型的检测方法，该检测方法用于生产用于兽医护理的类毒素。我们估计，我们的检测方法可以取代在药品开发和验证过程中使用数千只小鼠进行不道德的测试。BoNT/B敏感细胞系的可用性和BoNT/B活性的定量测定将有助于开发用于治疗常见神经障碍的新的改进的治疗剂。

Botulinum neurotoxins (BoNTs) are the most potent toxins known, with serotypes A, B, E causing human botulism and B, C, D causing animal botulism. Produced by Clostridium bacteria, BoNTs are highly lethal. However, in controlled conditions, some BoNTs variations (serotypes) can be used in a number of medical applications, such as neuromuscular spasm treatments. The main aim of this project is to establish a world-leading, quantitative in-vitro assay for detection of BoNT type B activity. This will allow replacement of the mouse lethality test currently used in production of pharmaceutical products related to BoNT type B. With previous support from the NC3Rs, we genetically engineered the first cell line that is sensitive to BoNT/B. We also incorporated a luminescent reporter system allowing convenient detection of BoNT/B activity using a user friendly microplate assay. Now, using our cell line and in partnership with the National Institute of Biological Standards and Control (NIBSC), we propose to both increase the sensitivity of the cell line and to develop a monoclonal antibody-based BoNT/B activity assay that outperforms the mouse bioassay on both specificity and sensitivity. Validation of our assay will be performed using pharmaceutical BoNT/B preparations and BoNT/B antitoxins. The successful implementation of this project will provide a firm incentive for BoNT/B manufacturers to replace the mouse LD50 testing with a faster, cheaper and more specific cell-based approach. We will also apply our technology to engineer an assay for detection of BoNT type D which is used for production of toxoids for veterinary care. We estimate that our assay can replace the use of thousands of mice in unethical testing during development and validation of pharmaceutical products. The availability of a BoNT/B-sensitive cell line and the quantitative assay for BoNT/B activity will aid development of new improved therapeutics for treatment of common neurological disorders.