In vitro potency determination of botulinum neurotoxin B based on its receptor-binding and proteolytic characteristics. (August 2016)

• Record Type: Journal Article
• Authors: Wild, Emina; Bonifas, Ursula; Klimek, Jolanta; Trösemeier, Jan-Hendrik; Krämer, Beate; Kegel, Birgit; Behrensdorf-Nicol, Heike A.
• Is Part Of: Toxicology in vitro. Volume 34(2016)
• Page Start: 97
• Page End: 104
• Publication Date: 2016-08
• Subjects: BINACLE binding and cleavage assay -- BoNT botulinum neurotoxin -- BSA bovine serum albumin -- GT1b ganglioside GT1b -- H-chain heavy chain -- HCc C-terminal region of the H-chain -- L-chain light chain -- LD50 lethal dose for 50% of test animals -- NAP neurotoxin-associated protein -- PBS phosphate-buffered saline -- PIPES piperazine-N, N′-bis(2-ethanesulfonic acid) -- Syt synaptotagmin peptide -- TCEP tris(2-carboxyethyl)phosphine -- TMAO trimethylamine N-oxide -- TMB 3, 3′, 5, 5′-tetramethylbenzidine; Botulinum neurotoxin serotype B -- In vitro assay -- 3R -- Binding and cleavage ability -- Activity determination -- Synaptobrevin; Toxicity testing -- In vitro -- Periodicals; Toxicology -- Periodicals; 615.9
• DOI: 10.1016/j.tiv.2016.03.011
• Languages: English
• ISSNs: 0887-2333

Abstract

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 0.1 mouse LD50 /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. Highlights: The BoNT/B BINACLE assay can assess the specific activity and potency of BoNT/B. A receptor peptide and ganglioside GT1b are used to selectively bind BoNT/B. After binding, active BoNT/B is quantified based on its proteolytic activity. Less than 0.1 pg BoNT/B can be detected in a sample volume of 100 μl.