Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E
(a) Clone selection and passage stability of different clones. More than 20 clones isolated for the expression of the reporter. Clones 3A1, 3A4, 3A9, 3A14, 3A17, 3A21, 5A3, 5A5, and 5A9 were tested for the growth, performance, and stability over 20 passages and chosen for the assay development. Clones were frozen at each passage, and cells were thawed and grown for an additional passage before being subjected to the sensor assay protocol with 20 nM BoNT/A treatment. The data shown here is for passage 15. Emission ratios were calculated by dividing the emission at 585 nm (DsRED) by the emission at 475 nm (GFP) ratio compared to control experiments (no BoNT treatment). Data are presented as means ± SD (). (b) Two clones, 3A14 and 5A3, chosen for further subcloning and FRET studies due to their superior performance and stability for more than 20 passages and subjected to 0–10 nM BoNT/A. Emission ratios were calculated by dividing the emission at 585 nm (DsRED) by the emission at 475 nm (GFP). Data are presented as means ± SD ().