Department of Biological Sciences, Allergan Inc., Irvine, California, United States of America.
PLoS One. 2012;7(11):e49516. doi: 10.1371/journal.pone.0049516. Epub 2012 Nov 21.
Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.
A型肉毒毒素（BoNT/A）是一种用于治疗多种疾病的强效治疗药物，通过切割 SNAP25 来抑制囊泡神经递质的胞吐作用。由于其效力强大，开发用于评估 BoNT/A 生物学功能的基于细胞的效力测定法（CBPAs）一直具有挑战性。CBPAs 可以评估 BoNT 作用的关键步骤：受体结合、内化-易位和催化活性；因此可以替代当前的小鼠生物测定法。原代神经元具有适当的敏感性，可以开发潜在的替代测定法，但这些效力测定法难以执行和验证。本报告描述了一种利用分化的人神经母细胞瘤 SiMa 细胞和夹心 ELISA 的 CBPA，该 ELISA 可测量 BoNT/A 依赖性细胞内切割的 SNAP25 增加。测定法的灵敏度与小鼠生物测定法相似，可测量散装药物和 BOTOX®产品（onabotulinumtoxinA）中的神经毒素生物学活性。在质量控制实验室对该 CBPA 进行验证后，已获得美国 FDA、加拿大卫生部和欧盟批准，可用于 BOTOX®、BOTOX®Cosmetic 和 Vistabel®的效力测试。此外，我们还开发并优化了一种 BoNT/A CBPA 筛选测定法，可用于发现治疗人类疾病的新型 BoNT/A 抑制剂。
