Horseradish peroxidase hrp conjugated anti human igg antibody

High-binding 96 well plates (Corning, New York) were pre-coated with 10 μg/ml concanavalin A (Sigma), a plant lectin that binds to glycoproteins [31 (link)]. Wells were washed and then incubated with UV-inactivated ZIKV (1×10⁵ plaque forming unit (PFU) /well) overnight at 4°C. After blocking with 5% bovine serum albumin (Sigma) in PBS containing 0.05% Tween-20, serially diluted inactivated serum samples were added into plates for 1 h at room temperature. Reactions were visualized by incubation with horseradish peroxidase (HRP)-conjugated anti-human IgG antibody (Abcam), followed by adding tetramethylbenzidine (TMB) substrate (Life Technologies). The endpoint titer was defined as the reciprocal of the lowest serum dilution above two times the average OD450 values of wells without applying serum samples.

ELISA was performed according to published protocol^{21 (link)}. Polystyrene microplates (Corning) were coated overnight with 2 μg/mL SARS-CoV-2 RBD protein (Sino Biological). After washed with PBS containing 0.2% Tween 20 (Solarbio Life Sciences), the plates were blocked using 2% BSA (Sigma Aldrich) in PBST for 1 h at 37 °C. Following washing with PBST, serial dilutions of testing antibodies were added to each well and incubated at 37 °C for 1 h. After washing with PBST, horseradish peroxidase (HRP)-conjugated anti-human IgG antibody (Abcam) was added at the dilution of 1:10,000 and incubated at 37 °C for 1 h. After washing, TMB singlecomponent substrate solution (Solarbio Life Sciences) was added to the microplate and incubated at room temperature for 6 min, followed by adding 2 M H₂SO₄ to stop the reaction. The absorbance was detected at 450 nm/630 nm. The data was analyzed using GraphPad Prism 8.0.