Viral Architecture of SARS-CoV-2 with Post-Fusion Spike Revealed by Cryo-EM

Chuang Liu; Yang Yang; Yuanzhu Gao; Chenguang Shen; Bin Ju; Congcong Liu; Xian Tang; Jinli Wei; Xiaomin Ma; Weilong Liu; Shuman Xu; Yingxia Liu; Jing Yuan; Jing Wu; Zheng Liu; Zheng Zhang; Peiyi Wang; Lei Liu

Microtiter plates (Sangon Biotech) were coated overnight at 4°C with 2.5 × 10 3 TCID 50 /well/100 µl purified and inactivated SARS-CoV-2 particles. The plates were washed twice with PBS containing 0.1% v/v Tween-20 (PBST) and blocked with blocking solution (PBS containing 2% w/v non-fat dry milk) for 2 hours at 37°C. The plates were then washed with PBST. The plasma were diluted to 1000-fold and human monoclonal antibody specific to SARS-CoV-2-RBD generated by our laboratory were diluted to 200 µg/ml into PBS as initial concentration, and serial 3-fold dilutions of sera was added to the wells and incubated at 37°C for 60 minutes. After three washes, 100 µl of horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody solution (Sangon Biotech) was added to each well and incubated at 37°C for 60 minutes. After washing, 100 µl of tetramethylbenzidine (TMB) substrate (Sangon Biotech) was added at room temperature in the dark. After 15 minutes, the reaction was stopped with a 2M H2SO4 solution. The absorbance was measured at 450 nm. All samples were run in triplicate. The ELISA titers were determined by endpoint dilution.