Biotin rat anti mouse igg2a

96-well plate (Thermo Fisher Scientific, MA, USA) were coated with 5 µg/ml of WT, Beta, Delta, or Omicron RBD or spike protein at 4 °C overnight. Plates were then blocked with 3% skim-milk/PBS at room temperature for 2 h. Serum samples were serially diluted and added to the blocked plates before incubation at room temperature for an hour. Following incubation, bound antibodies were either detected with goat anti-mouse IgG Fc HRP-conjugated antibody (Chemicon, CA, USA) for total IgG assessment or biotin-rat-anti-mouse IgG1 (BD Biosciences, NJ, USA) and biotin-rat-anti-mouse IgG2a (BD Biosciences, NJ, USA), and then followed by HRP- streptavidin (R&D Systems, MN, USA) for IgG subclass assessment. Plates were developed by TMB substrate (BD Biosciences, NJ, USA) and the reactions were stopped by adding 2N H₂SO₄. The absorbance at 450 nm were measured with EMax Microplate reader (Molecular Devices, CA, USA). The endpoint dilution titer was determined when titer value of the last serum dilution was twofold above the blank value.