Table 1. Assay Optimization and Standardization.

Parameter	Conditions tested	Optimal
96-well culture plate	7 matrices	Poly-D-Lysine
Differentiation medium	8 formulations	EMEM SFM plus N2 & B27 supplements and GT1b
Differentiation time	6 time points	≥48 h
Seeding cell density	4 densities	50,000–100,000 cells/well
Treatment medium	5 formulations	Differentiation medium without GT1b
BoNT/A treatment time	3 time points	24 h
Incubation time	4 incubation times	2 days
BoNT/A dose range	0.004–300 pM	0.004 pM (S/B = 4.5)–25 pM
Amount of capture and detection antibodies	24 combinations	2E2A6 spotted 5 µL at 20 µg/mL Detection (S9684) 25 µL at 5 µg/mL
Lysate incubation	3 temperatures and time	Overnight at 4°C
Blocking Buffers	3 buffers	2% ECL blocking with 10% goat serum
Detection antibody	2 temperatures plus time	1 h at room temperature
Edge Effects	Outside rows and columns	Edge effects. Avoid rows A and H; columns 1 and 12