Table 1.

Design of experiment factors and levels investigated for DEN2

Factor	Whole plot or subplot	Level 1	Level 2	Level 3	Level 4
A. Plate incubation temperature	Whole plot	Ambient	37°C	N/A	N/A
B. Primary Ab incubation time and temperature	Whole plot	60 minutes/37°C	Overnight/4°C	N/A	N/A
C. Secondary Ab incubation time	Whole plot	30 minutes	90 minutes	N/A	N/A
D. Cell seeding density	Subplot	1 × 104 cells	3 × 104 cells	N/A	N/A
E. Multiplicity of infection	Subplot	0.01	0.1	N/A	N/A
F. Neutralization mix incubation-time/temp	Subplot	30 minutes/37°C	Overnight/4°C	60 minutes/37°C	60 minutes/ambient
G. Primary Ab concentration	Subplot	1 µg/mL	10 µg/mL	N/A	N/A
H. Secondary Ab dilution	Subplot	1:250	1:1,000	N/A	N/A

N/A = not applicable.