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  • Title: Assessment of ELISA as endpoint in neuronal cell-based assay for BoNT detection using hiPSC derived neurons.
    Author: Pellett S, Tepp WH, Johnson EA, Sesardic D.
    Journal: J Pharmacol Toxicol Methods; 2017 Nov; 88(Pt 1):1-6. PubMed ID: 28465161.
    Abstract:
    INTRODUCTION: 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. METHODS: 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. RESULTS: 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). DISCUSSION: 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.
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