Analytical and Bioanalytical Chemistry

, Volume 406, Issue 28, pp 7149–7161 | Cite as

Validation of the Endopep-MS method for qualitative detection of active botulinum neurotoxins in human and chicken serum

  • Kristian Björnstad
  • Annica Tevell Åberg
  • Suzanne R. Kalb
  • Dongxia Wang
  • John R. Barr
  • Ulf Bondesson
  • Mikael HedelandEmail author
Research Paper


Botulinum neurotoxins (BoNTs) are highly toxic proteases produced by anaerobic bacteria. Traditionally, a mouse bioassay (MBA) has been used for detection of BoNTs, but for a long time, laboratories have worked with alternative methods for their detection. One of the most promising in vitro methods is a combination of an enzymatic and mass spectrometric assay called Endopep-MS. However, no comprehensive validation of the method has been presented. The main purpose of this work was to perform a validation for the qualitative analysis of BoNT-A, B, C, C/D, D, D/C, and F in serum. The limit of detection (LOD), selectivity, precision, stability in matrix and solution, and correlation with the MBA were evaluated. The LOD was equal to or even better than that of the MBA for BoNT-A, B, D/C, E, and F. Furthermore, Endopep-MS was for the first time successfully used to differentiate between BoNT-C and D and their mosaics C/D and D/C by different combinations of antibodies and target peptides. In addition, sequential antibody capture was presented as a new way to multiplex the method when only a small sample volume is available. In the comparison with the MBA, all the samples analyzed were positive for BoNT-C/D with both methods. These results indicate that the Endopep-MS method is a valid alternative to the MBA as the gold standard for BoNT detection based on its sensitivity, selectivity, and speed and that it does not require experimental animals.


Botulinum neurotoxin Endopep-MS Method validation Botulism MALDI-Q-TOF 



The authors would like to thank Dr. James Marks and Dr. Jianlong Lou at the University of California San Francisco for the help in selecting antibodies suitable for this study. We would also like to thank Hanna Skarin at SVA for providing PCR data and C. botulinum cultivation broths. The financial support from the framework of the EU-project AniBioThreat (Grant Agreement: Home/2009/ISECAG/191), the Prevention of and Fight against Crime Programme of the European Union, European Commission–Directorate General Home Affairs, and the Swedish Civil Contingencies Agency (MSB) are greatly acknowledged. This publication reflects the views only of the authors, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Kristian Björnstad
    • 1
  • Annica Tevell Åberg
    • 1
    • 2
  • Suzanne R. Kalb
    • 3
  • Dongxia Wang
    • 3
  • John R. Barr
    • 3
  • Ulf Bondesson
    • 1
    • 2
  • Mikael Hedeland
    • 1
    • 2
    Email author
  1. 1.Department of Chemistry, Environment and Feed HygieneNational Veterinary Institute (SVA)UppsalaSweden
  2. 2.Division of Analytical Pharmaceutical ChemistryUppsala UniversityUppsalaSweden
  3. 3.Division of Laboratory Sciences, National Center for Environmental HealthCenters for Disease Control and PreventionAtlantaUSA

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