Caliskan, C., Simsek, D., Leese, C. orcid.org/0000-0002-3123-0154 et al. (4 more authors) (2024) A sensitive cell-based assay for testing potency of Botulinum neurotoxin type A. ALTEX, 41 (4). pp. 605-616. ISSN 1868-596X
Abstract
Botulinum neurotoxin type A (BoNT/A) is a biopharmaceutic widely used for the treatment of neurological diseases and in aesthetic medicine to achieve months-long paralysis of target muscles and glands. Large numbers of mice are used in the mouse bioassay (MBA) for various botulinum-related applications including batch release potency testing, antitoxin testing, countermeasure development, and basic research. BoNT/A intoxication causes severe suffering to the mice used for testing, and application-specific, non-animal alternatives are urgently needed. Cell-based assays (CBA) need to replicate all the physiological steps of botulinum intoxication, comprising neuronal binding, internalization, endosomal escape, and cleavage of synaptosomal-associated protein of 25 kDa (SNAP25). However, the CBA currently in use have limitations. In this study we show that LAN5 cells, a human neuroblastoma-derived cell line, are sensitive to BoNT/A and can be engineered to express a recombinant NanoLuciferase (NanoLuc)-tagged SNAP25 reporter molecule. On exposure, the reporter molecule is cleaved and releases a NanoLuc-SNAP25 fragment that can be captured on a 96-well plate for quantitative luminometry using a cleavage-specific SNAP25 antibody. We demonstrate, using purified BoNT/A and a commercial BoNT/A product, that the sensitivity of this new cell-based assay is in the fM range, comparable to that of the MBA. The new assay could replace the MBA in research and commercial testing of BoNT/A, benefiting both scientific progress and animal welfare.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © The Authors, 2024. This work is licensed under a Creative Commons Attribution 4.0 International License. (https://creativecommons.org/licenses/by/4.0/) |
Keywords: | LAN5; NanoLuc; SNAP-25; botulinum neurotoxin; cell assay; Botulinum Toxins, Type A; Humans; Animal Testing Alternatives; Biological Assay; Animals; Synaptosomal-Associated Protein 25; Mice; Cell Line, Tumor |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) |
Funding Information: | Funder Grant number BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL BB/W017210/1 BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL BB/L002841/1 BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL BB/S009566/1 |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 29 Jan 2025 15:30 |
Last Modified: | 29 Jan 2025 15:30 |
Status: | Published |
Publisher: | ALTEX Edition |
Refereed: | Yes |
Identification Number: | 10.14573/altex.2312071 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:222429 |