Detection of biologically active botulinum neurotoxin-A in serum using high-throughput FRET-assay

Abstract

IntroductionThe goals of this project were to compare fluorescent resonance energy transfer (FRET) assays using a customized FRET substrate (substrate–substrate-A, SSA) with a commercially available FRET substrate (SNAPtide); optimize the assay conditions for SSA for lowest level of detection; and apply SSA to detect botulinum neurotoxin-A (BoNTA) in serum samples. MethodsBiological activity of BoNTA and light-chain-A (LCA) was verified by murine phrenic nerve-hemidiaphragm bioassay and western blot before use in both FRET assays. The reaction conditions were optimized to determine the smallest amount of toxin that could be detected. A range of serum samples was investigated for interference in the SSA-based FRET assay. Detection of BoNTA from rat serum samples was performed over time. ResultsWe found that BoNTA and LCA were able to cleave the substrates whereas mutated LCA and a different serotype of BoNT, BoNTB, could not. SSA had significantly more arbitrary fluorescing units compared to the FRET substrate SNAPTide, and the SSA assay could detect 0.1nM of BoNTA or LCA comfortably (p=<0.05) in a 20-μl reaction. No significant interference was observed when serum was present in the reaction buffer. Due to negligible background noise, the SSA FRET assay could detect BoNTA from spiked rat serum even after 256min. DiscussionThe greatest advantage of the FRET assay is its extreme rapidity, its cost effectiveness, and unlike ELISA, its ability to detect biologically active toxin. SSA is a better FRET substrate for detecting BoNTA toxin (detected 0.1nM concentration). Because serum present in the assay reaction did not cause any appreciable interference, the assay can be used to detect BoNTA in serum samples. Therefore, the SSA FRET assay can be used for pharmacokinetic and pharmacodynamic studies, screening inhibitors, and detecting BoNTA in serum samples.