A highly sensitive replacement assay for botulinum neurotoxin type B

肉毒杆菌神经毒素（BoNTs）是已知最有效的毒素，血清型A、B、E引起人类肉毒中毒，血清型B、C、D引起动物肉毒中毒。bont由梭状芽胞杆菌产生，具有高度致命性。然而，在受控条件下，一些bont变异（血清型）可用于许多医学应用，如神经肌肉痉挛治疗。该项目的主要目的是建立一种世界领先的体外定量检测B型BoNT活性的方法。这将取代目前用于生产与B型BoNT相关的药物产品的小鼠致死试验。在NC3Rs之前的支持下，我们对第一个对BoNT/B敏感的细胞系进行了基因工程改造。我们还结合了一个发光报告系统，允许使用用户友好的微孔板试验方便地检测BoNT/B活性。现在，利用我们的细胞系，并与美国国家生物标准与控制研究所（NIBSC）合作，我们建议提高细胞系的敏感性，并开发一种基于单克隆抗体的BoNT/B活性测定方法，该方法在特异性和敏感性上都优于小鼠生物测定方法。我们的检测将使用药物BoNT/B制剂和BoNT/B抗毒素进行验证。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LD50测试。我们还将应用我们的技术设计一种检测BoNT D型的检测方法，用于生产兽医护理用的类毒素。我们估计，我们的分析可以取代在药品开发和验证过程中使用数千只老鼠进行不道德的测试。BoNT/B敏感细胞系的可用性和BoNT/B活性的定量分析将有助于开发用于治疗常见神经系统疾病的新改进疗法。

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.