title: A simple, rapid and sensitive FRET assay for botulinum neurotoxin serotype B detection.
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.
authors.affiliation: Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR.
authors.forename: Jiubiao
authors.initials: J
authors.lastname: Guo
authors.affiliation: Department of Chemistry, University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Ci
authors.initials: C
authors.lastname: Xu
authors.affiliation: Department of Chemistry, University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Xuechen
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR.
authors.forename: Sheng
authors.initials: S
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D019274:Botulinum Toxins, Type A / Q000032:analysis* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D050988:Vesicle-Associated Membrane Protein 2 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D050988:Vesicle-Associated Membrane Protein 2; C096323:rimabotulinumtoxinB; D019274:Botulinum Toxins, Type A