title: Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters.
abstract: Methods that do not require animal sacrifice to detect botulinum neurotoxins (BoNTs) are critical for BoNT antagonist discovery and the advancement of quantitative assays for biodefense and pharmaceutical applications. Here we describe the development and optimization of fluorogenic reporters that detect the proteolytic activity of BoNT/A, B, D, E, F, and G serotypes in real time with femtomolar to picomolar sensitivity. Notably, the reporters can detect femtomolar concentrations of BoNT/A in 4h and BoNT/E in 20h, sensitivity that equals that of animal-based methods. The reporters can be used to determine the specific activity of BoNT preparations with intra- and inter-assay coefficients of variation of approximately 10%. Finally, we find that the greater sensitivity of our reporters compared with those used in other commercially available assays makes the former attractive candidates for high-throughput screening of BoNT antagonists.
authors.affiliation: BioSentinel Pharmaceuticals, Madison, WI 53719, USA.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Ruge
authors.forename: F Mark
authors.initials: FM
authors.lastname: Dunning
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Piazza
authors.forename: Brian E
authors.initials: BE
authors.lastname: Molles
authors.forename: Michael
authors.initials: M
authors.lastname: Adler
authors.forename: Füsûn N
authors.initials: FN
authors.lastname: Zeytin
authors.forename: Ward C
authors.initials: WC
authors.lastname: Tucker
mesh_terms: D001905:Botulinum Toxins / Q000032:analysis* / Q000145:classification; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D017930:Genes, Reporter; D007700:Kinetics; D012703:Serotyping; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature
chemical_list: D005456:Fluorescent Dyes; D001905:Botulinum Toxins