Pellett Sabine, Tepp William H, Johnson Eric A, Sesardic Dorothea
Department of Bacteriology, University of Wisconsin, 1550 Linden Dr., Madison, WI 53706, United States.
Division of Bacteriology, National Institute for Biological Standards and Control (NIBSC), A centre of Medicines and Healthcare products Regulatory Agency; Hertfordshire EN6 3QG, UK.
J Pharmacol Toxicol Methods. 2017 Nov;88(Pt 1):1-6. doi: 10.1016/j.vascn.2017.04.013. Epub 2017 Apr 29.
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 (EC50~0.3U/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.
肉毒杆菌神经毒素（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的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，唯一被批准的用于测定药用BoNTs效力的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基于细胞的测定法能够替代MBA用于特定产品评估。本项目详细介绍了一种使用酶联免疫吸附测定（ELISA）来定量和灵敏地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，该方法用于确定毒素暴露后BoNT/A1对SNAP-25的切割情况。
将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，在细胞裂解物中评估和优化毒素活性的定量检测。
本研究结果表明，使用超敏TMB作为底物的ELISA能够定量检测BoNT/A1暴露的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0.3U/孔）。
本研究展示了一种基于人特异性且灵敏的细胞检测平台，用于检测BoNT/A1活性，该平台以ELISA作为终点来定量检测SNAP-25切割产物。该测定法适用于中高通量形式，重要的是采用非癌性人特异性神经元细胞来评估一种供人使用的生物药物的效力。
