The proposed in-vitro assay aims to replace use of animals in the manufacture of pharmaceutical BoNT/B products. BoNT/B product testing involves the most severe levels of animal suffering due to death by slow asphyxiation being the assay endpoint. The LD50 assay also lacks specificity to distinguish between the different BoNTs serotypes, which all cause similar muscular paralysis.
With previous NC3Rs support, we engineered a human neuroblastoma SiMa cell line to carry a synthetic VAMP reporter molecule, the target of BoNT/B. We also developed a polyclonal antibody which recognises the BoNT/B-cleaved end of the VAMP reporter molecule. By selecting right capture plates we were able to design a refined ELISA capture assay which can detect the BoNT/B-cleaved VAMP via highly sensitive luminescent reaction. This advance was published in Frontiers in Pharmacology in 2017.
To build a robust 3Rs legacy we must now create an assay for easy assimilation into a GMP-compliant environment, allowing widespread adoption by BoNT/B product manufacturers and regulators. In order to achieve this, we must produce monoclonal antibodies that can specifically detect VAMP2 cleaved by BoNT/B and devise a microplate-based assay for reproducible sensitive detection of BoNT/B activity. To make our ELISA assay most compelling, we will improve the sensitivity of our cell lines to further outperform the mouse bioassay by introducing the receptor with the highest known affinity for BoNT/B, mouse synaptotagmin 2 into our engineered cell lines. We will then screen for the most sensitive clones using industrial and in-house pharmaceutical BoNT/B and related BoNT/D. Finally, in partnership with our collaborators we will validate our highly specific and reproducible assay for suitability to test botulinum antitoxins and other pharmaceutical products.
Gimenez-Molina Y et al. (2019). Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells. Scientific Reports 9:18471. doi: 10.1038/s41598-019-55106-w