Potency testing of Botulinum toxin and antitoxin therapies is entirely dependent on mouse lethality bioassay where groups of mice are exposed to the toxin by intra-peritoneal injection and monitored over 72 or 96 hours. Due to a lethal endpoint and the large number of laboratory animals used, the mouse LD50 assay is considered inhumane and new alternative methods are required to refine, replace and to reduce animal use. More humane non-lethal local flaccid paralysis assays have been developed which are approximately 10 times more sensitive than the lethality method but still require large numbers of animals. The use of primary cells is limited because of the continued requirement for animals, time-consuming dissections and potential contamination with non-neuronal components.
One approach to avoid these limitations is to develop sensitive human cell line models. To date, cell based approaches using established cell lines have failed to provide a sensitive functional endpoint and new human cell based assays are still eagerly required in new therapy strategies. In order to develop a suitable human cell model, human neuroblastoma cell lines have been established in culture at NIBSC and routinely differentiated into mature neurons. The suitability of differentiated SH-SY5Y cells for detecting high picomolar levels of Botulinum type A has already been evidenced using neurotransmitter release measurements.
Application of micro-electrode arrays (MEAs) platform as a potential functional neurotoxicity assay will be implemented in parallel with imaging of synaptic vesicle recycling, immunodetection and neurotransmitter release measurements. This new approach is entirely non animal orientated and will not rely on any animal experimentation widely used in other applications and strategies. In vitro functional neurotoxicity assays offers the potential of entire animal replacement because of its mode of action involving all key steps of Botulinum intoxication.
Bak N et al. (2017). SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E. Toxins 9(7):230. doi: 10.3390/toxins9070230
Sesardic T (2012). Bioassays for evaluation of medical products derived from bacterial toxins. Current Opinion in Microbiology 15(3):310-316. doi: 10.1016/j.mib.2012.05.008
Rasetti-Escargueil C et al. (2011). Enhanced sensitivity to Botulinum type A neurotoxin of human neuroblastoma SH-SY5Y cells after differentiation into mature neuronal cells. The Botulinum Journal 2(1):30-48. doi: 10.1504/TBJ.2011.041814
- Research Review 2013: New assays for botulinum toxin testing
Principal investigatorDr Dorothea Sesardic
InstitutionNational Institute for Biological Standards and Control
Co-InvestigatorDr Christine Escargueil
Dr Roland Fleck