Cytation 7 microplate multi mode reader

SARS-CoV-2 pseudoviruses were generated as described below.¹⁵ (link)^,26 (link)^,27 (link)^,28 (link) Specifically, plasmid encoding S protein of the original strain or respective variant of SARS-CoV-2 was transfected into 293T cells in the presence of pLenti-CMV-luciferase plasmid and PS-PAX2 plasmid (Addgene) using the polyetherimide (PEI) (Sigma) transfection assay. 72 h after the transfection, pseudovirus-containing supernatants were collected, and used for pseudovirus neutralization assay. Each pseudovirus was incubated with serially diluted mouse sera at 37°C for 1 h, which was added to hACE2/293T cells; 24 h later, fresh medium was added to the cells, and the cells were cultured at 37°C in the presence of 5% CO₂, followed by lysis using cell lysis buffer (Promega) 48 later. The supernatant of lysed cells was incubated with luciferase substrate (Luciferase Assay System) (Promega), which was then measured for relative luciferase activity using Cytation 7 Microplate Multi-Mode Reader and Gen5 software (BioTek Instruments). Pseudovirus neutralizing activity was reported as 50% neutralizing antibody titer (NT₅₀).

Enzyme-linked immunoassay (ELISA) was used to measure specific serum antibodies from immunized mice.¹⁵ (link)^,26 (link) Specifically, 96-well ELISA plates were precoated with each recombinant SARS-CoV-2 S or RBD protein (1 μg/mL)²⁶ (link) at 4°C overnight, and blocked with blocking buffer (e.g., 2% fat-free milk dissolved in PBST (0.05% Tween-20 in PBS)) at 37°C for 1 h. The plates were then incubated with serially diluted mouse sera at 37°C for 1 h, and washed with PBST for at least three times. This step was followed by further incubation of the plates with horseradish peroxidase (HRP)-conjugated anti-mouse IgG-Fab (1:5,000, Sigma), anti-mouse IgG1, anti-mouse IgG2a, and anti-mouse IgG2b (1:5,000, Invitrogen) antibodies, respectively, at 37°C for 1 h, and washing for three times. After incubation of the plates with 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate (Sigma), the reaction was stopped by addition of 1 N H₂SO₄. The absorbance at 450 nm was measured using Cytation 7 Microplate Multi-Mode Reader and Gen5 software (BioTek Instruments).