ZIKV E Protein Antibody Detection

The ZIKV E protein-specific antibodies in sera were detected using an ELISA kit (Alpha Diagnostic, USA) according to the manufacturer’s instructions. Briefly, 100 μl aliquots of diluted serum (1:50,000) were added per well. Z_EDIII-specific antibodies (IgG, IgG1, and IgG2c) in sera were determined by indirect ELISA as described previously [26 (link), 27 (link)]. Microplates (Nunc-Immuno Plates; Thermo Scientific, UK) were coated with Z_EDIII (1 μg/ml) at 4 °C overnight. Each well was washed with 0.05% Tween 20 in PBS (PBST) and then with 1% BSA in PBS and incubated at 37 °C for 2 h. The plates were washed and then diluted serum was incubated at 37 °C for 2 h. Plates were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG heavy and light chain antibody (1:10,000; Bethyl Laboratories, TX, USA), goat anti-mouse IgG1 (1:10,000; Bethyl Laboratories), or goat anti-mouse IgG2c (1:8000; Southern Biotech, USA) at 37 °C for 1 h and washed with PBST. Color development was performed using tetramethylbenzidine substrate (Surmodics, USA) and stopped with 2 N H₂SO₄. The optical density of the plates was read at 450 nm in an ELISA plate reader (BioTek, Winooski, NT, USA).