SARS-CoV-2 Nucleocapsid Antibody ELISA

SARS-CoV-2 nucleocapsid-specific antibodies were detected by ELISA as in our previously reported method [26 (link),27 (link)]. In brief, 96-well EIA plates (Corning Inc, Corning, NY, USA) were coated with 100 ng per well of SARS-CoV-2 N protein antigens (Abclonal, Wuhan, China) in PBS overnight at 4 °C. Plates were washed with PBST three times and then blocked with 5% skimmed milk in PBST for 1 h at 37 °C. Mouse serum was diluted at 1:25, and then added to the washed plates and incubated for 2 h at 37 °C. After washing, plates were incubated with a 1:5000 dilution of HRP conjugated anti-mouse IgG secondary antibody (Abcam, Cambridge, UK), and HRP conjugated anti-mouse IgG1 secondary antibody (Abcam), HRP conjugated anti-mouse IgG2a secondary antibody (Proteintech, Wuhan, China), and HRP conjugated anti-mouse IgG2c secondary antibody (Abcam, Cambridge, UK), respectively, for 1 h at 37 °C. The color reaction was substrated with 3,3′,5,5′-Tetramethylbenzidine (TMB) and stopped with 1 M H₂SO₄. The value was read at a 450 nm wavelength using a Synergy HT Multi-Mode Plate Reader (BioTek, Winooski, VT, USA).