SARS-CoV-2 Spike Protein ELISA Assay

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.