Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters |
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| Authors: | Ruge Daniel R Dunning F Mark Piazza Timothy M Molles Brian E Adler Michael Zeytin Füsûn N Tucker Ward C |
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| Institution: | aBioSentinel Pharmaceuticals, Madison, WI 53719, USA;bDepartment of Forest and Wildlife Ecology, University of Wisconsin–Madison, Madison, WI 53706, USA;cNeurobehavioral Toxicology Branch, US Army Medical Research Institute of Chemical Defense (USAMRICD), Aberdeen Proving Ground–Edgewood Area (APG–EA), MD 21010, USA |
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| 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 4 h and BoNT/E in 20 h, 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. |
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| Keywords: | Botulinum neurotoxin Proteases FRET High-throughput assays |
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