Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay
Figure 4
SiMa cells are sensitive and specific for BoNT/A uptake.
A. Differentiated SiMa cells were treated with 1 nM BoNT/A complex from 1 to 60 min. Cell lysates were evaluated in the ECL-sandwich ELISA. Signal above background was observed at the earliest time points indicating high affinity binding (Error bars = std. dev.). B. Specificity of uptake by SiMa cells was demonstrated by comparing the uptake of BoNT/A (150 kDa) to recombinant LHN/A (lacking binding domain but comprising the translocation domain and an active light chain) and inactive BoNT/A (iBoNT/A, inactive light chain). No cleavage of SNAP25 was detected with iBoNT/A. Non-specific uptake of LHN/A was observed only at doses >10 nM. C. Graph comparing specific uptake of BoNT/A complex (at pM concentrations) to a full dose-response of recombinant LHN/A (at pM to µM concentrations) with a highest dose of 50 µM. The EC50 for the LHN/A molecule was 2.1 µM versus 0.85 pM for the fully active BoNT/A.