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1.
Toxicol In Vitro ; 53: 80-88, 2018 Dec.
Article in English | MEDLINE | ID: mdl-30016653

ABSTRACT

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.


Subject(s)
Botulinum Toxins, Type A/toxicity , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Neurotoxins/toxicity , Peptides/metabolism , Animal Testing Alternatives , Animals , Biological Assay , Mice , Protein Binding , Proteolysis , Recombinant Proteins/metabolism , Synaptosomal-Associated Protein 25/metabolism
2.
Toxicol In Vitro ; 34: 97-104, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27032463

ABSTRACT

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.


Subject(s)
Botulinum Toxins, Type A/metabolism , Gangliosides/metabolism , Synaptotagmin II/metabolism , Vesicle-Associated Membrane Protein 2/metabolism , Biological Assay , Protein Binding , Proteolysis
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