Tmb substrate

Recombinant SARS-CoV-2 Spike protein (50 ng, Sino Biological) diluted in carbonate buffer (0.1 M, pH 9.6) were coated into 96-well EIA/RIA plates (Coning) overnight at 4 °C. The plates were then washed with PBS-T (0.05% Tween-20) and were blocked with 10% goat serum in PBS for 2 h at 37 °C. Then, serum samples serially diluted in PBST containing 2% goat serum were added and incubated for 2 h at 37 °C. After washing, total IgG was evaluated using HRP-conjugated goat anti-mouse IgG Ab (1:10,000) or HRP-conjugated goat anti-NHP IgG Ab (1:50,000) for 1 h. In mice experiments, IgG subclasses were evaluated using biotinylated anti-mouse IgG2a mAb (clone: MG2a, Mabtech), biotinylated anti-mouse IgG1 mAb (clone: MG1, Mabtech), biotinylated anti-mouse IgG2c mAb (clone: MTG2c, Mabtech) for 1 h and further analyzed with Streptavidin-HRP (1:1000, Mabtech). TMB substrate (Solarbio) was used for development and the absorbance was read at 450 nm using SPECTROsta Nano (BMG) microplate reader. Endpoint titers were calculated as the dilution that exceed 2.1-folded value of the background.

High‐binding 96‐Well ELISA plates were coated with OVA protein (20 µg mL⁻¹) in a Na₂CO₃/NaHCO₃ buffer (pH = 9.6) at 4 °C overnight. After being washed with 0.05% tween‐PBS solution, the plates were blocked with 3% BSA solution. The antiserum was then diluted to different concentrations using 1% BSA in PBS and added to the plates (100 µL well⁻¹) and incubated for 2 h at 37 °C. The plates were washed for three times, and incubated with rabbit anti‐mouse IgG‐Peroxidase antibodies (SBA, 1:2000 dilution) for 1 h at 37 °C. Then the plates were further washed for three times, followed by the addition of TMB substrate (Solarbio), and reacted at room temperature for 20 min in dark. The corresponding OD₄₅₀ was measured by Multiskan GO (Thermo Scientific) microplate reader.

An ELISA assay assessing binding antibody levels against the wild-type SARS-CoV-2 was conducted using the wild-type Spike protein. ELISA plates (Corning Costar; Corning, NY, USA) were coated with S protein (Sanyou Biopharmaceuticals Co., Shanghai, China) at a concentration of 5 μg/well and incubated overnight at 4 °C. During the experiment, ELISA plates were blocked with 5% BSA-phosphate-buffered saline (PBS), incubated with serially diluted serum samples, and visualized by reaction with an HRP-conjugated antibody (Abcam, Waltham, MA, USA) and TMB substrate (Solarbio, Beijing, China), using previously described methods [16 (link)]. The absorbance of each well at 450 nm was measured using an ELISA plate reader (Gene Company, Beijing, China). Antibody serum samples yielding OD values at least 2.1-fold higher than the negative control at a test sample dilution of 1:400 were considered positive. Endpoint titers (ETs) were defined as the highest serum dilutions yielding positive OD values. GMTs were calculated as the geometric mean of the ETs of positive serum samples in each group. ELISA assays assessing binding antibody levels against variant strains of SARS-CoV-2 (Alpha, Beta, Gamma, Delta, and Omicron) were conducted using commercial ELISA kits (ACRO Biosystems Co., Beijing, China) according to the manufacturer’s instructions.