Sars cov 2 nucleocapsid antibody

MA104 cells and Vero E6 cells were seeded in 96-well plates and used when reached close to 100% confluency. Harvested supernatants from infected cells were serial diluted and inoculated onto prepared 96-well plates with MA104 cells or Vero E6 cells. After 1 h incubation at 37°C, the inoculum was removed and replaced with fresh media and the plate was incubated at 37°C overnight for 16–18 h. Cells infected with VSV-SARS-CoV-2 was directly taken under the microscope and examined for GFP. Cells infected with replication-competent SARS-CoV-2 WA1 was fixed with 4% PFA for 15 min, washed twice with PBS, and permeabilized with 0.1% Triton-X100 in PBS for 10 min. After two washes with PBS with 0.1% Tween-20 (PBST), the cells were blocked for 1 h at room temperature using 1%BSA with 10%FBS in PBST. Cells were incubated at 4°C overnight with SARS-CoV-2 nucleocapsid antibody (Sino Biological, 40588-T62) in PBST +1% BSA, diluted at 1:1,000. Post primary antibody incubation, cells were washed twice in PBST and incubated for 45 min in dark with anti-rabbit IgG HRP-linked antibody (Cell Signalling, 7074S) in PBST +1% BSA, diluted at 1:1,000. Finally, the cells were incubated with AEC substrate (Vector Laboratories, SK-4200) for antigen labeling.