SARS-CoV-2 Spike Protein Antibody Titers

Anti-spike antibody titers were measured using ELISA as previously described^{19 (link)}. Briefly, 96-well plates were coated with 1 μg/ml recombinant spike protein (ancestral spike; SPN-C52H9, Omicron BA.1 spike; SPN-C52Hz, Omicron BA.2 spike; SPN-C5223, Omicron BA.5 spike; SPN-C522e, Acro Biosystems) and kept at 4 ℃ overnight. After blocking with 5% skim milk PBS for 2 h at RT, the diluted sera (ranging from 10- to 31,250-fold dilution with 5% skim milk PBS) were added to the wells and incubated at 4 °C overnight. The following day, the wells were washed with PBS-T and incubated with HRP-conjugated anti-IgG antibody (GE Healthcare) for 3 h at RT. The wells were developed using the peroxidase chromogenic substrate 3,3′-5,5′-tetramethyl benzidine (TMB, Sigma) for 30 min at RT. The reaction was quenched by adding sulfuric acid (0.5 N). The absorbance of the samples in the wells was measured immediately at 450 nm using a microplate reader (Bio-Rad). The half-maximum antibody titer of the sample was determined from the highest absorbance in the dilution range (GraphPad Prism 8 software). For IgG subclass determination, anti-IgG1 (Abcam), IgG2a (Abcam), IgG2b (Abcam), and IgG2c (Southern Biotech) antibodies were used as the secondary antibodies.