Indirect ELISA

Purified CVA6-W of 1×104 TCID50 were diluted with the ELISA coating buffer (pH 9.6) (Solarbio, China) and added into each well of 96-well ELISA plate (Costar, USA). All plates were stored at 4°C temperature overnight and then washed with PBST (pH 7.2–7.4) (Solarbio, China). Then, 100 µl blocking buffer (5% bovine serum albumin [BSA] in PBST) (Solarbio, China) were added into each well and stored at 37°C for 1 h. After the plate was washed, the 10-fold serially diluted antisera in blocking buffer were added into the plate and incubated at 37°C for 1 h. The plates were washed three times with PBST and incubated with a 1:8,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG(H+L) (CWBIO, China) secondary antibody or HRP-conjugated goat anti-mouse IgM (Abcam, UK) at 37°C for 1 h. After the plate was washed four times, 100 µl TMB Single-Component Substrate solution (Solarbio, China) were added into each well of the plates for 10 min. Then 100 µl ELISA Stop Solution (Solarbio, China) were added into each well and the results were analyzed with an ELISA plate reader (Multiskan MK3; Thermo Scientific) at 450 nm.