FIG. 7.

BoNT/A enzymatic assay optimization using PL50 and PL51. BoNT/A was incubated at increasing concentrations at 37°C with either 10 μM PL50 (▪) or 10 μM PL51 (▾) under optimized conditions. Substrate stock solutions were prepared so as to obtain a final DMF concentration of 1.5% in 100 μl of the optimized reaction buffer (HEPES, 20 mM, pH 7.4; ZnSO₄, 200 μM; TCEP, 2.5 mM). The inset represents a zoom of the lower BoNT/A concentrations, which allowed us to determine the detection limits, namely, 100 pg (53 MLD₅₀s) or 60 pg (32 MLD₅₀s) in 120 min using PL50 or PL51, respectively. Mean values of endpoint measurements (120 min) from two independent experiments performed in duplicate are represented.