title: Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test.
abstract: The detection and identification of botulinum neurotoxins (BoNT) is complex due to the existence of seven serotypes, derived mosaic toxins and more than 40 subtypes. Expert laboratories currently use different technical approaches to detect, identify and quantify BoNT, but due to the lack of (certified) reference materials, analytical results can hardly be compared. In this study, the six BoNT/A1-F1 prototypes were successfully produced by recombinant techniques, facilitating handling, as well as improving purity, yield, reproducibility and biosafety. All six BoNTs were quantitatively nicked into active di-chain toxins linked by a disulfide bridge. The materials were thoroughly characterized with respect to purity, identity, protein concentration, catalytic and biological activities. For BoNT/A₁, B₁ and E₁, serotypes pathogenic to humans, the catalytic activity and the precise protein concentration were determined by Endopep-mass spectrometry and validated amino acid analysis, respectively. In addition, BoNT/A₁, B₁, E₁ and F₁ were successfully detected by immunological assays, unambiguously identified by mass spectrometric-based methods, and their specific activities were assigned by the mouse LD50 bioassay. The potencies of all six BoNT/A1-F1 were quantified by the ex vivo mouse phrenic nerve hemidiaphragm assay, allowing a direct comparison. In conclusion, highly pure recombinant BoNT reference materials were produced, thoroughly characterized and employed as spiking material in a worldwide BoNT proficiency test organized by the EQuATox consortium.
authors.affiliation: Toxogen GmbH, Feodor-Lynen-Str. 35, 30625 Hannover, Germany. weisemann@toxogen.de.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Weisemann
authors.affiliation: Toxogen GmbH, Feodor-Lynen-Str. 35, 30625 Hannover, Germany. krez@toxogen.de.
authors.forename: Nadja
authors.initials: N
authors.lastname: Krez
authors.affiliation: Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany. fiebigu@rki.de.
authors.forename: Uwe
authors.initials: U
authors.lastname: Fiebig
authors.affiliation: Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany. worbss@rki.de.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Worbs
authors.affiliation: Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany. skibam@rki.de.
authors.forename: Martin
authors.initials: M
authors.lastname: Skiba
authors.affiliation: Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany. tanja.endermann@ayoxxa.com.
authors.forename: Tanja
authors.initials: T
authors.lastname: Endermann
authors.affiliation: Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany. dornerm@rki.de.
authors.forename: Martin B
authors.initials: MB
authors.lastname: Dorner
authors.affiliation: Division of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Cementvägen 20, 90182 Umeå, Sweden. tomas.bergstrom@foi.se.
authors.forename: Tomas
authors.initials: T
authors.lastname: Bergström
authors.affiliation: Joint Research Centre, Institute for Reference Materials and Measurements, European Commission, Retieseweg 111, 2440 Geel, Belgium. amalia.munoz-pineiro@ec.europa.eu.
authors.forename: Amalia
authors.initials: A
authors.lastname: Muñoz
authors.affiliation: Joint Research Centre, Institute for Reference Materials and Measurements, European Commission, Retieseweg 111, 2440 Geel, Belgium. ingrid.zegers@ec.europa.eu.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Zegers
authors.affiliation: Federal Department of Defence, Civil Protection and Sport-Spiez Laboratory, Austrasse 1, 3700 Spiez, Switzerland. christian.mueller@babs.admin.ch.
authors.forename: Christian
authors.initials: C
authors.lastname: Müller
authors.affiliation: Federal Department of Defence, Civil Protection and Sport-Spiez Laboratory, Austrasse 1, 3700 Spiez, Switzerland. stephen.jenkinson@ifik.unibe.ch.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Jenkinson
authors.affiliation: Federal Department of Defence, Civil Protection and Sport-Spiez Laboratory, Austrasse 1, 3700 Spiez, Switzerland. marc-andre.avondet@babs.admin.ch.
authors.forename: Marc-Andre
authors.initials: MA
authors.lastname: Avondet
authors.affiliation: Scientific Service of Food-Borne Pathogens, Operational Directorate of Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), 1050 Brussels, Belgium. laurence.delbrassinne@wiv-isp.be.
authors.forename: Laurence
authors.initials: L
authors.lastname: Delbrassinne
authors.affiliation: Scientific Service of Food-Borne Pathogens, Operational Directorate of Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), 1050 Brussels, Belgium. sarah.denayer@wiv-isp.be.
authors.forename: Sarah
authors.initials: S
authors.lastname: Denayer
authors.affiliation: Joint Research Centre, Institute for Reference Materials and Measurements, European Commission, Retieseweg 111, 2440 Geel, Belgium. reinhard.zeleny@ec.europa.eu.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Zeleny
authors.affiliation: Joint Research Centre, Institute for Reference Materials and Measurements, European Commission, Retieseweg 111, 2440 Geel, Belgium. heinz.schimmel@ec.europa.eu.
authors.forename: Heinz
authors.initials: H
authors.lastname: Schimmel
authors.affiliation: Division of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Cementvägen 20, 90182 Umeå, Sweden. crister.astot@foi.se.
authors.forename: Crister
authors.initials: C
authors.lastname: Åstot
authors.affiliation: Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany. dornerb@rki.de.
authors.forename: Brigitte G
authors.initials: BG
authors.lastname: Dorner
authors.affiliation: Toxogen GmbH, Feodor-Lynen-Str. 35, 30625 Hannover, Germany. rummel@toxogen.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Rummel
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000032:analysis* / Q000737:chemistry / Q000633:toxicity; D005260:Female; D059021:Laboratory Proficiency Testing / Q000592:standards; D007928:Lethal Dose 50; D051379:Mice; D009498:Neurotoxins / Q000032:analysis* / Q000737:chemistry / Q000633:toxicity; D010791:Phrenic Nerve / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000633:toxicity; D012015:Reference Standards; D050600:SNARE Proteins / Q000737:chemistry
chemical_list: D009498:Neurotoxins; D011994:Recombinant Proteins; D050600:SNARE Proteins; D001905:Botulinum Toxins
keywords: botulinum neurotoxin; characterization; mass spectrometric identification; recombinant expression; reference material; specific activity