Abstract
Tetanus neurotoxin (TeNT) consists of two protein chains connected by a disulfide linkage: The heavy chain mediates the toxin binding and uptake by neurons, whereas the light chain cleaves synaptobrevin and thus blocks neurotransmitter release.Chemically inactivated TeNT (tetanus toxoid) is utilized for the production of tetanus vaccines. For safety reasons, each toxoid bulk has to be tested for the "absence of toxin and irreversibility of toxoid". To date, these mandatory tests are performed as toxicity tests in guinea pigs. A replacement by an animal-free method for the detection of TeNT would be desirable. The BINACLE (BINding And CLEavage) assay takes into account the receptor-binding as well as the proteolytic characteristics of TeNT: The toxin is bound to immobilized receptor molecules, the light chains are then released by reduction and transferred to a microplate containing synaptobrevin, and the fragment resulting from TeNT-induced cleavage is finally detected. This assay offers a higher specificity for discriminating between toxic TeNT and inactivated toxoid molecules than other published assays. Validation studies have shown that the BINACLE assay allows the sensitive and robust detection of TeNT in toxoids, and thus may indeed represent a suitable alternative to the prescribed animal safety tests for toxoids from several European vaccine manufacturers. Product-specific validations (and possibly adaptations) of the assay protocol will be required. A European collaborative study is currently being initiated to further examine the applicability of the method for toxoid testing. The final aim is the inclusion of the method into the European Pharmacopoeia.
Highlights
This article describes the “BINACLE” (BINding And CLEavage) assay for in vitro detection of active tetanus neurotoxin
Each toxoid bulk has to be tested for the “absence of toxin and irreversibility of toxoid” according to the instructions of the European Pharmacopoeia before it can be used in vaccines for human or veterinary use (Council of Europe, 2014a,b)
This study showed that the BINACLE assay is able to detect active TeNT with a detection limit of approximately 0.03 ng/ml, which is comparable to the estimated detection limit of the in vivo test
Summary
This article describes the “BINACLE” (BINding And CLEavage) assay for in vitro detection of active tetanus neurotoxin. It provides an overview of the current state of assay validation and highlights the potential applicability of this new assay for the safety and consistency testing of tetanus vaccines. The toxoids contain a large variety of molecular constructs bearing diverse intra- or inter-molecular crosslinks. These formaldehyde-induced modifications are thought to directly block or impede the native function of the involved protein domains (like, e.g., the receptor binding or the proteolytic domain). Inter-chain crosslinks may contribute to detoxification by preventing the release of the light toxin chain into the neuronal cytosol (Thaysen-Andersen et al, 2007)
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