Assessment of ELISA as endpoint in neuronal cell-based assay for BoNT detection using hiPSC derived neurons.

Botulinum neurotoxins (BoNTs), the causative agents of botulism, are widely used as powerful bio-pharmaceuticals to treat neuro-muscular disorders. Due to the high potency and potential lethality of BoNTs, careful monitoring of the biologic activity of BoNT-based pharmaceuticals is required to ensure safe usage. For decades, the only approved method for potency determination of pharmaceutical BoNTs was the mouse bioassay (MBA), but in recent years improvements in cell-assay technologies have enabled MBA replacement by cell-based assays for specific product evaluations. This project details a method for quantitative and sensitive detection of biologic activity of BoNT/A1 in human induced pluripotent stem cell (hiPSC) derived neurons using an ELISA as a method to determine SNAP-25 cleavage by BoNT/A1 following toxin exposure. HiPSC derived neurons from two different sources were exposed to serial dilutions of BoNT/A1, and quantitative detection of toxin activity was evaluated and optimized in cell lysates using ELISA to detect cleaved SNAP-25. The results from this study indicate that an ELISA using ultra TMB as a substrate quantitatively detects cleaved SNAP-25 in cell lysates of BoNT/A1 exposed hiPSC-derived neuronal cells with similar or greater sensitivity as Western blot (EC50 ~ 0.3 U / well). This study demonstrates a human specific and sensitive cell-based detection platform of BoNT/A1 activity using ELISA as an endpoint for quantitative detection of the SNAP-25 cleavage product. This assay is applicable to moderate to high-throughput formats and importantly employs non-cancerous human-specific neuronal cells for potency evaluation of a bio-pharmaceutical for human use.

肉毒神经毒素（BoNTs）是肉毒中毒的病原体，被广泛用作治疗神经肌肉疾病的强效生物药物。由于BoNTs具有高效力和潜在致死性，需要仔细监测基于BoNT的药物的生物活性以确保安全使用。几十年来，测定药用BoNTs效力的唯一批准方法是小鼠生物测定法（MBA），但近年来细胞测定技术的改进使得在特定产品评估中能够用基于细胞的测定法取代MBA。本项目详细介绍了一种定量和灵敏检测人诱导多能干细胞（hiPSC）衍生神经元中BoNT/A1生物活性的方法，该方法使用酶联免疫吸附测定（ELISA）作为在毒素暴露后确定BoNT/A1对SNAP - 25切割的方法。 来自两种不同来源的hiPSC衍生神经元暴露于BoNT/A1的系列稀释液中，并使用ELISA检测切割后的SNAP - 25，对细胞裂解物中毒素活性的定量检测进行了评估和优化。 这项研究的结果表明，一种使用超灵敏TMB作为底物的ELISA能够定量检测暴露于BoNT/A1的hiPSC衍生神经元细胞裂解物中切割后的SNAP - 25，其灵敏度与蛋白质印迹法相似或更高（半数有效浓度约为0.3单位/孔）。 这项研究展示了一个基于人类特异性敏感细胞的BoNT/A1活性检测平台，使用ELISA作为对SNAP - 25切割产物进行定量检测的终点。该测定适用于中到高通量形式，重要的是使用非癌性的人类特异性神经元细胞来评估供人类使用的生物药物的效力。