title: In vitro potency determination of botulinum neurotoxin B based on its receptor-binding and proteolytic characteristics.
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.
authors.affiliation: Veterinary Medicine Division, Paul-Ehrlich-Institut (Federal Institute for Vaccines and Biomedicals), Langen, Germany.
authors.forename: Emina
authors.initials: E
authors.lastname: Wild
authors.affiliation: Veterinary Medicine Division, Paul-Ehrlich-Institut (Federal Institute for Vaccines and Biomedicals), Langen, Germany.
authors.forename: Ursula
authors.initials: U
authors.lastname: Bonifas
authors.affiliation: Veterinary Medicine Division, Paul-Ehrlich-Institut (Federal Institute for Vaccines and Biomedicals), Langen, Germany.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Klimek
authors.affiliation: Microbiology Division, Paul-Ehrlich-Institut (Federal Institute for Vaccines and Biomedicals), Langen, Germany; Molecular Bioinformatics, Institute of Computer Science, Johann Wolfgang Goethe University, Frankfurt am Main, Germany.
authors.forename: Jan-Hendrik
authors.initials: JH
authors.lastname: Trösemeier
authors.affiliation: Veterinary Medicine Division, Paul-Ehrlich-Institut (Federal Institute for Vaccines and Biomedicals), Langen, Germany.
authors.forename: Beate
authors.initials: B
authors.lastname: Krämer
authors.affiliation: Veterinary Medicine Division, Paul-Ehrlich-Institut (Federal Institute for Vaccines and Biomedicals), Langen, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Kegel
authors.affiliation: Veterinary Medicine Division, Paul-Ehrlich-Institut (Federal Institute for Vaccines and Biomedicals), Langen, Germany. Electronic address: Heike.Behrensdorf@pei.de.
authors.forename: Heike A
authors.initials: HA
authors.lastname: Behrensdorf-Nicol
mesh_terms: D001681:Biological Assay; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D005732:Gangliosides / Q000378:metabolism*; D011485:Protein Binding; D059748:Proteolysis; D050861:Synaptotagmin II / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism*
chemical_list: D005732:Gangliosides; D050861:Synaptotagmin II; C494263:Syt2 protein, mouse; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2; C096323:rimabotulinumtoxinB; C016481:trisialoganglioside GT1; D019274:Botulinum Toxins, Type A
keywords: 3R; Activity determination; Binding and cleavage ability; Botulinum neurotoxin serotype B; In vitro assay; Synaptobrevin