ELISPOT Assay for EV-A71-Specific Antibody Response

According to our previous study (35 (link)), Enzyme-linked immunospot (ELISPOT) was used to detect EV-A71-specific IgG- and IgA-secreting cells. We coated 96-well microplates (Millipore, United States) with purified EV-A71 virions (10 μg/ml) overnight and blocked the wells with 3% BSA in PBS. Then, spleen cells (5 × 10⁵) in RPMI-10% FBS were added and incubated overnight at 37°C. Following incubation, the plate was washed, and HRP-conjugated goat anti-mouse IgGs (1:000, Bethyl Laboratories, Inc.) or IgAs (1:000, Bethyl Laboratories, Inc.) was added. After incubating plates for 2 h, the HRP conjugate was removed and washed. Then, 100 μl of 3-amino-9-ethylcarbazole substrate (Sigma) was added and spots were allowed to develop in the dark at room temperature (RT) for 10 min. After ELISPOT analysis was completed, the plate was analyzed using an ImmunoSpot^® S6 UV Reader (Cellular Technology Limited, Cleveland, OH, United States). ImmunoSpot^® v.6.0 Software was used to automatically count the spots for each EV-A71 antigen stimulation condition and medium negative controls. Spots from a total of three wells with 1.5 × 10⁶ spleen cells were determined and considered as one set of data.