Product: Botulinum Neurotoxin Type E, Nicked, from Clostridium botulinum
… Nicked BoNT/E3 was obtained from List Biological Laboratories (Campbell, CA, USA). …
Sandwich Assay Procedure:
When performing the sandwich assay, for every mAb, the unlabeled mAb was used as the capture antibody and the biotinylated form was used as the detector antibody. Each mAb was tested in combination with all of the mAbs to identify pairs that can detect the BoNT/E3 toxin in the solution phase. Black plates were coated with 100 L of mAb at 2 g/mL in 0.05 M sodium carbonate buffer, pH 9.6, incubated overnight at 4 C, and then blocked with 3% NFDM-TBST. The blocking solution was removed and BoNT/E3 toxin in 3% NFDM-TBST was added at an initial concentration of 500 ng/mL and serially diluted two-fold, including wells with no toxin. Next, biotinylated-mAb was added in triplicate at 1 g/mL, 100 µL/well in 3% NFDM-TBST. Plates were incubated for 1 h at 37°C, washed 6 with TBST, and visualized using SuperSignal West Dura Extended Duration Substrate as described above.
When the most sensitive combination of capture mAb and detector mAb-biotin were selected, the final sandwich assays were performed in triplicate using BoNT/E3 holotoxin at a starting concentration of 1000 ng/mL, serially diluted three-fold, including wells with no toxin, and visualized using SuperSignal Femto Max Sensitivity substrate. Limit of detection cut-off values were determined using three times the standard deviation of the wells with no toxin.