Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E
(a) Morphology of PC12 cells acquired under bright field microscopy (×40) cultured according to standard methods. (b) Sensitivity of PC-12 cell lines to BoNT/A as shown by SNAP-25 cleavage efficiency. Western blot image of PC-12 cells lysate after treatment with BoNT/A. The cleavage product marked by an arrow. (c) Immunofluorescence staining of methanol-fixed PC-12 cells showing SNAP-25 membrane localization. Image was acquired using a Nikon Eclipse Ti-E confocal microscope (Nikon Inc., USA). (d) Schematic representation of the BoNT sensor constructs. A fusion protein construct with full-length SNAP-25 as a linker between AcGFP1 and DsRED-monomer was created resulting in the AcGFP-SNAP-25-DsRed construct. Arrows indicate the site of cleavage by BoNT/A, -C, -E.