Detection of serum antibodies
The IgG antibody titers of serum samples were analyzed by an indirect enzyme-linked immunosorbent assay (ELISA). Purified ZIKV E protein was diluted and added to 96-well microtiter plates (Corning-Costar, Corning, NY, USA) at 0.5 µg/well. After incubation at 4°C overnight and washing with PBST (PBS with 0.5% tween-20), the plates were blocked with 5% nonfat milk. Then, serum diluted 128-fold with blocking buffer was added, followed by HRP-conjugated goat anti-mouse IgG (H+L), IgG1 or IgG2a (1:2000). Tetramethylbenzidine (TMB) was added, and 2 M H2SO4 was used to stop color development. Absorbance was read at an optical density (OD) of 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The results are displayed as the positive index (ratio of sample OD450 to control OD450).
A virus neutralization assay (VNA) of ZIKV was performed as follows. The serum was serially diluted twofold and mixed with an equal volume of virus (50 PFU/100 μl) at 37°C for 1 h. Then, 200 μl of the serum-virus mixture was transferred to a monolayer of baby hamster kidney (BHK) cells. After culturing for 1 h, the mixture was replaced with a 2% agarose overlay. The cells were cultured for another 4 days at 37℃. After fixation with paraformaldehyde and staining with crystal violet, a standard 50% plaque reduction neutralization test (PRNT50) was performed to calculate the titer.