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

• Published in: Toxicology in vitro Vol. 53; pp. 80 - 88
• Main Authors: Behrensdorf-Nicol, Heike A., Wild, Emina, Bonifas, Ursula, Klimek, Jolanta, Hanschmann, Kay-Martin, Krämer, Beate, Kegel, Birgit
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.12.2018; Elsevier Science Ltd
• Subjects: Acetylcholine; Activity determination; Assaying; Binding; Binding and cleavage assay (BINACLE assay); Botulinum neurotoxin serotype A (BoNT/A); Botulinum toxin; Botulinum toxin type A; Chains; Chemical reduction; Cleavage; In vitro assay; Lethal dose; Motor neurons; Muscle contraction; Muscles; Neurons; Neurotoxicity; Neurotoxins; Neurotransmitters; Organic chemistry; Proteins; Proteolysis; SNAP-25 protein; Substrates; Test procedures; Toxicity; Toxins; PBS; TMAO; L-chain; BoNT; LD50; TeNT; In vitro assay; BSA; H-chain; SV2C; BINACLE; HCcA; NAP; Botulinum neurotoxin serotype A (BoNT/A); TCEP; Binding and cleavage assay (BINACLE assay); Activity determination; GT1b
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/j.tiv.2018.07.008

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. •The BoNT/A BINACLE assay can assess the specific activity and potency of BoNT/A.•A SV2C-derived peptide and ganglioside GT1b are used to specifically bind BoNT/A.•After binding, active BoNT/A is quantified based on its SNAP-25-cleaving activity.•<0.05 mouse LD50 units of BoNT/A can be detected in a sample of 100 μl.