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Toxins 2015, 7(12), 5035-5054; doi:10.3390/toxins7124861

Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test

1
Toxogen GmbH, Feodor-Lynen-Str. 35, 30625 Hannover, Germany
2
Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany
3
Division of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Cementvägen 20, 90182 Umeå, Sweden
4
Joint Research Centre, Institute for Reference Materials and Measurements, European Commission, Retieseweg 111, 2440 Geel, Belgium
5
Scientific Service of Food-Borne Pathogens, Operational Directorate of Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), 1050 Brussels, Belgium
6
Federal Department of Defence, Civil Protection and Sport—Spiez Laboratory, Austrasse 1, 3700 Spiez, Switzerland
*
Author to whom correspondence should be addressed.
Academic Editor: Xiaohua He
Received: 15 October 2015 / Revised: 10 November 2015 / Accepted: 13 November 2015 / Published: 26 November 2015
View Full-Text   |   Download PDF [2007 KB, uploaded 26 November 2015]   |  

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. View Full-Text
Keywords: botulinum neurotoxin; reference material; recombinant expression; specific activity;characterization; mass spectrometric identification botulinum neurotoxin; reference material; recombinant expression; specific activity;characterization; mass spectrometric identification
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Weisemann, J.; Krez, N.; Fiebig, U.; Worbs, S.; Skiba, M.; Endermann, T.; Dorner, M.B.; Bergström, T.; Muñoz, A.; Zegers, I.; Müller, C.; Jenkinson, S.P.; Avondet, M.-A.; Delbrassinne, L.; Denayer, S.; Zeleny, R.; Schimmel, H.; Åstot, C.; Dorner, B.G.; Rummel, A. Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test. Toxins 2015, 7, 5035-5054.

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