Transferability study of the BINACLE (binding and cleavage) assay for in vitro determination of botulinum neurotoxin activity.

The muscle-relaxing effects of the botulinum neurotoxin (BoNT) serotypes A and B are widely used in clinical and aesthetic medicine. The standard method for measuring the biological activity of pharmaceutical BoNT products is a mouse bioassay. In line with the European Directive 2010/63/EU, a replacement by an animal-free method would be desirable. Whereas the existing approved in vitro methods for BoNT activity measurements are product-specific and not freely available for all users, the "binding and cleavage" (BINACLE) assay could become a widely applicable alternative. This method quantifies active BoNT molecules based on their specific receptor-binding and proteolytic properties and can be applied to all BoNT products on the European market. Here we describe the results of a transferability study, in which identical BoNT samples were tested in the BINACLE assay in four laboratories. All participants successfully performed the method and observed clear dose-response relationships. Assay variability was within an acceptable range. These data indicate that the BoNT BINACLE assay is robust and can be straightforwardly transferred between laboratories. They thus provide an appropriate basis for future studies to further substantiate the suitability of the BINACLE assay for the potency determination of BoNT products.

In vitro potency determination of botulinum neurotoxin serotype A based on its receptor-binding and proteolytic characteristics.

Botulinum neurotoxins (BoNTs) inhibit the release of the neurotransmitter acetylcholine from motor neurons, resulting in highly effective muscle relaxation. In clinical and aesthetic medicine, serotype BoNT/A, which is most potent for humans, is widely used to treat a continuously increasing spectrum of disorders associated with muscle overactivity. Because of the high toxicity associated with BoNTs, it is mandatory to precisely determine the potency of every batch produced for pharmaceutical purposes. Here we report a new quantitative functional in vitro assay for BoNT/A. In this binding and cleavage (BINACLE) assay, the toxin is first bound to specific receptor molecules. Then a chemical reduction is performed, thereby releasing the light chain of BoNT/A and activating its proteolytic domain. The activated light chain is finally exposed to its substrate protein SNAP-25, and the fragment resulting from the proteolytic cleavage of this protein is quantified in an antibody-mediated reaction. The BoNT/A BINACLE assay offers high specificity and sensitivity with a detection limit below 0.5 mouse lethal dose (LD50)/ml. In conclusion, this new in vitro assay for determining BoNT/A toxicity represents an alternative to the LD50 test in mice, which is the "gold standard" method for the potency testing of BoNT/A products.

In vitro potency determination of botulinum neurotoxin B based on its receptor-binding and proteolytic characteristics.

Botulinum neurotoxins (BoNTs) are the most potent toxins known. However, the paralytic effect caused by BoNT serotypes A and B is taken advantage of to treat different forms of dystonia and in cosmetic procedures. Due to the increasing areas of application, the demand for BoNTs A and B is rising steadily. Because of the high toxicity, it is mandatory to precisely determine the potency of every produced BoNT batch, which is usually accomplished by performing toxicity testing (LD50 test) in mice. Here we describe an alternative in vitro assay for the potency determination of the BoNT serotype B. In this assay, the toxin is first bound to its specific receptor molecules. After the proteolytic subunit of the toxin has been released and activated by chemical reduction, it is exposed to synaptobrevin, its substrate protein. Finally the proteolytic cleavage is quantified by an antibody-mediated detection of the neoepitope, reaching a detection limit below 0.1mouseLD50/ml. Thus, the assay, named BoNT/B binding and cleavage assay (BoNT/B BINACLE), takes into account the binding as well as the protease function of the toxin, thereby measuring its biological activity.

In vitro determination of tetanus toxicity by an endopeptidase assay linked to a ganglioside-binding step.

Assays for the detection of tetanus neurotoxin (TeNT) are relevant for research applications as well as for the safety testing of tetanus vaccines. So far, these assays are usually performed as toxicity tests in guinea pigs or mice. The alternative methods described to date were mostly based on the detection of the toxin's proteolytic activity. However, these endopeptidase assays turned out to be unreliable because they only measure the enzymatic activity as sole determinant of tetanus toxicity, while not taking into account other parameters like the toxin's capacity to bind to target cells. In order to better reflect the in vivo situation of a tetanus infection, we have linked an endopeptidase assay to a ganglioside-binding step. The resulting method, which offers a unique combination of two functionally linked assays, detects those TeNT molecules only which possess both a functional binding domain as well as an active enzymatic domain. Our results demonstrate that this assay is able to reliably detect TeNT, and therefore might provide a basis for the replacement of the animal tests for detection of tetanus toxicity. Moreover, the assay concept could also be useful for in vitro toxicity measurements of other toxins with similar subunit structures.