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Antibodies and Vaccines against Botulinum Toxins: Available Measures and Novel Approaches
Open AccessArticle

Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments

1
Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany
2
Institut für Toxikologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
*
Author to whom correspondence should be addressed.
Toxins 2019, 11(10), 588; https://doi.org/10.3390/toxins11100588
Received: 31 July 2019 / Revised: 1 October 2019 / Accepted: 9 October 2019 / Published: 11 October 2019
(This article belongs to the Special Issue Characterization and Quantitative Analysis of Botulinum Neurotoxin)
The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins’ light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A–F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A–F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays. View Full-Text
Keywords: botulinum neurotoxins; detection; substrate cleavage; taguchi design-of-experiments; SNARE botulinum neurotoxins; detection; substrate cleavage; taguchi design-of-experiments; SNARE
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von Berg, L.; Stern, D.; Weisemann, J.; Rummel, A.; Dorner, M.B.; Dorner, B.G. Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments. Toxins 2019, 11, 588.

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