Development of cell-based assays for testing Clostridial Neurotoxins

Clostridial neurotoxins are protein structures generated by a bacterium. Clostridial neurotoxins consist of botulinum neurotoxin, with its serotypes, and tetanus neurotoxin. A small amount of these proteins is lethal as they cause muscle paralysis. However, safe doses are used for cosmetic and medical purposes. Due to their potency, it is essential to develop a sensitive assay to detect small amounts of the neurotoxins for several reasons. Firstly, safe doses should be determined before releasing them as medical or cosmetic treatments. Secondly, it is important to test food products in order to prevent food poisoning. Thirdly, neurotoxins could be used as a bioweapon, so detection of small amounts is necessary for security. Currently, the mouse bioassay is the international gold standard test. However, it has many drawbacks. The test is expensive, time-consuming, requires advanced facilities and skills, and, most importantly, raises ethical issues in relation to animal welfare. Approximately 100 mice are killed in every test and experience significant suffering. Therefore, developing a fast, cheap, and sensitive replacement assay which adheres to 3Rs (Replace, Refine, and Reduce animals in research) principles is essential. A main aim of this project is to develop a replacement assay for sensitive detection of clostridial neurotoxins to fulfil this gap. In this project, neuroblastoma cells are mainly used to test clostridial toxin activity, and this involves 3Rs principles. Several neuroblastoma cell lines are generated through the project to get better results for toxin sensitivity. The neurotoxins are first tested by immunoblotting whether the toxin could cleave target proteins in neuroblastoma cell lines. However, a one-step ELISA method is developed to measure toxin level in a faster and more practical way. Immunocytochemistry and immunoblotting techniques are used to characterise neuroblastoma cell lines to confirm why they are sensitive to the clostridial neurotoxins. It was found that neuroblastoma cell lines are quite sensitive for testing most of the clostridial neurotoxins. In particular, BoNT/A, BoNT/B, BoNT/C, BoNT/DC, and TeNT activities could be measured quickly and sensitively in neuroblastoma cell lines. Neuroblastoma cells are promising for developing a cell-based assay to replace the mouse assay for most of the clostridial neurotoxins. The cell-based assay developed in this research is quite sensitive, however there is still room to improve sensitivity. Moreover, this assay allows for the detection of all steps of clostridial intoxication: Toxin-receptor binding, translocation of toxin inside cells and SNARE cleavage by toxin. Previous studies use SiMa human neuroblastoma cell line for botulinum neurotoxin A and botulinum neurotoxin B activities. However, this research has introduced a new sensitive neuroblastoma cell line for clostridial neurotoxins, the LAN-5 human neuroblastoma cell. My research has contributed more knowledge about the relationship between neuroblastoma cells and clostridial neurotoxins. I have also introduced other cell type, HeLa cells, for BoNT/B potency. To conclude, this PhD study discovered new sensitive cell lines to test clostridial neurotoxin potency. This is promising for the development of sensitive replacement cell-based assays.