A highly sensitive replacement assay for botulinum neurotoxin type B

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Botulinum neurotoxins (BoNTs) are the most potent toxins known, with serotypes A, B, E causing human botulism and B, C, D causing animal botulism. Produced by Clostridium bacteria, BoNTs are highly lethal. However, in controlled conditions, some BoNTs variations (serotypes) can be used in a number of medical applications, such as neuromuscular spasm treatments. The main aim of this project is to establish a world-leading, quantitative in-vitro assay for detection of BoNT type B activity. This will allow replacement of the mouse lethality test currently used in production of pharmaceutical products related to BoNT type B. With previous support from the NC3Rs, we genetically engineered the first cell line that is sensitive to BoNT/B. We also incorporated a luminescent reporter system allowing convenient detection of BoNT/B activity using a user friendly microplate assay. Now, using our cell line and in partnership with the National Institute of Biological Standards and Control (NIBSC), we propose to both increase the sensitivity of the cell line and to develop a monoclonal antibody-based BoNT/B activity assay that outperforms the mouse bioassay on both specificity and sensitivity. Validation of our assay will be performed using pharmaceutical BoNT/B preparations and BoNT/B antitoxins. The successful implementation of this project will provide a firm incentive for BoNT/B manufacturers to replace the mouse LD50 testing with a faster, cheaper and more specific cell-based approach. We will also apply our technology to engineer an assay for detection of BoNT type D which is used for production of toxoids for veterinary care. We estimate that our assay can replace the use of thousands of mice in unethical testing during development and validation of pharmaceutical products. The availability of a BoNT/B-sensitive cell line and the quantitative assay for BoNT/B activity will aid development of new improved therapeutics for treatment of common neurological disorders.