Sars cov 2 s

To assess the antibody response against the different SARS-CoV-2 proteins or different fragments of the spike protein, SARS-CoV-2 S (spike protein, 1203 aa), S1 (675 aa), S2 (533 aa), RBD (228 aa) and N (424 aa) proteins were obtained from Sino Biological, Inc (Beijing) and used as coating antigens in our in-house ELISA to detect the antigen-specific IgG. Briefly, 96-well plates (Jet, Biofil Co., Ltd, Guangzhou) were coated with 100 μl/well (0.5 μg/ml) of SARS-CoV-2 S, S1, S2, RBD or N protein in DPBS buffer (Thermo Fisher Scientific (China), Shanghai) overnight at 4°C. After blocking (DPBS, 10%FBS), 100 μl of diluted plasma (1:100) were added and plates were incubated at 37°C for one hour. After washing, plates were incubated with 100 μl of HRP-conjugated mouse anti-human IgG (H+L) antibody (Catalog No: 109-035-088, Jackson ImmunoResearch, West Grove, PA) at 37°C for one hour. Reactions were visualized by adding 50 μl of TMB (3,3’,5,5’-Tetramethylbenzidine) substrate solution (Biohao Biotechnology Co., Ltd., Beijing). Each plate was monitored to have the same reaction time and the optical densities at 450 nm were then read. The mean value of the healthy donor plasma (named HD group) collected in 2017-2018 plus 3 standard deviations was used as the detection threshold.

Serum samples from SARS-CoV-2 infected animals were inactivated by γ-irradiation and used in BSL2 according to IBC-approved SOPs. NUNC Maxisorp Immuno plates were coated with 50 μl of 1 μg/mL of recombinant SARS-CoV-2 S (S1+S2), SARS-CoV-2 RBD (Sino Biological) or EBOV GP (32 (link)) at 4°C overnight and then washed three times with PBS containing 0.05% Tween 20 (PBST). The plates were blocked with 3% skim milk in PBS for 3 hours at room temperature, followed by three additional washes with PBST. The plates were incubated with 50 μl of serial dilutions of the samples in PBS containing 1% skim milk for 1 hour at room temperature. After three washes with PBST, the bound antibodies were labeled using 50 μl of 1:2,500 peroxidase anti-hamster IgG (H+L) (SeraCare Life Sciences) diluted in 1% skim milk in PBST. After incubation for 1 h at room temperature and three washes with PBST, 50 μl of KPL ABTS peroxidase substrate solution mix (SeraCare Life Sciences) was added to each well, and the mixture was incubated for 30 min at room temperature. The optical density (OD) at 405 nm was measured using a GloMax^® explorer (Promega) plate reader. The OD values were normalized to the baseline samples obtained with naïve hamster serum and the cutoff value was set as the mean OD plus standard deviation of the blank.