Goat anti mouse igg1

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).

The sera were tested for ZIKV E protein-specific antibodies using commercially available ELISA kits (Alpha Diagnostic, USA) according to the manufacturer’s instructions. Briefly, 100 μl aliquots of diluted serum (1:50,000) were added per well. Env_M- or Env_Z-specific antibodies (IgG, IgG1, and IgG2c) in sera were determined by indirect ELISA. Microplates (Nunc-Immuno Plates; Thermo Scientific, UK) were coated with Env_M (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).

100 µL of peripheral blood was collected 1 week before vaccination and 15 days post-vaccination. Blood was left to clot at 4 °C for 5 h. Serum samples were obtained by centrifugation at 8,000 xg for 10 min and stored at -20 °C until use. Serial dilutions (1/3) of sera were prepared with 1% BSA in PBS-0.05% Tween 20. 96-well flat-bottom microplates were coated with 50 μL of 50 μg/mL recombinant S1+S2 ECD-His protein (Sino Biological, Beijing, China) and RBD (RayBiotech Life, Peachtree Corners, GA) in 3.36 mM carbonate–10 mM bicarbonate buffer at 4° C for 16 h. After three washes with 1% BSA in PBS-0.05% Tween 20, blocking was performed in PBS-0.05% Tween 20 containing 3% BSA for 1 h at room temperature. Next, the washes were repeated, and 100 μL of diluted serum samples were added, incubating for 1 h. After repeating the wash step, incubations with 1:8,000-diluted HRP-conjugated protein A (Invitrogen, Waltham, MA), 1:20,000 goat anti-mouse IgG1, or 1:20,000 goat anti-mouse IgG2c (Bethyl Laboratories, Montgomery, TX) were performed for 1 h, followed by three final washes. Color development was executed with TMB Substrate Kit (Thermo Scientific, Waltham, MA) for 10 min in 100 µL. The reactions were stopped by adding 100 μL of 2 N H₂SO₄. A₄₅₀ was registered with Microplate Reader 680 (BioRad, Hercules, CA) and Microplate Manager 5.2.1 software (BioRad, Hercules, CA).