A simple, rapid and sensitive FRET assay for botulinum neurotoxin serotype B detection

  • PLoS One. 2014 Dec 1;9(12):e114124. doi: 10.1371/journal.pone.0114124.
Jiubiao Guo  1 ,  Ci Xu  2 ,  Xuechen Li  2 ,  Sheng Chen  1
Affiliations
  • 1. Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR.
  • 2. Department of Chemistry, University of Hong Kong, Pokfulam, Hong Kong.
PMID: 25437190 DOI: 10.1371/journal.pone.0114124
Abstract

Botulinum neurotoxins (BoNTs), the most potent naturally-occurring neurotoxins known to humans, comprise seven distinct serotypes (BoNT/A-G), each of which exhibits unique substrate specificity. Many methods have been developed for BoNT detection, in particular for BoNT/A, with various complexity and sensitivity, while substrate based FRET assay is considered as the most widely used approach due to its simplicity and sensitivity. In this study, we designed a vesicle-associated membrane protein 2 (VAMP2) based FRET assay based on the understanding of the VAMP2 and light chain/B (LC/B) interactions in our previous studies. The current design constituted the shortest peptide, VAMP2 (63-85), with FRET dyes (EDAN and Dabcyl) labelled at position 76 and 85, respectively, which showed minimal effect on VAMP2 substrate catalysis by LC/B and therefore enhanced the sensitivity of the assay. The FRET peptide, designated as FVP-B, was specific to LC/B, with a detection sensitivity as low as ∼20 pM in 2 h. Importantly, FVP-B showed the potential to be scaled up and used in high throughput screening of LC/B inhibitor. The currently developed FRET assay is one of the most economic and rapid FRET assays for LC/B detection.

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