Ensight luminometer

The Omicron variant gene of SARS-CoV-2 spike protein (GISAID: EPI_ISL_6590782.2) was optimized using mammalian codon and synthesized, then cloned into pcDNA3.1 vector as described previously [11 (link)]. The plasmid expressing the S protein of 614G, Alpha, Beta, Gamma, Delta, Lambda, and Mu SARS-CoV-2 variants were previously constructed (Figures S1). The VSV-based pseudotyped SARS-CoV-2 variants was produced by transfecting 293T cells (American Type Culture Collection, CRL-3216) with S protein expression plasmids and simultaneously infected with G*ΔG-VSV (Kerafast, Boston, MA). The titres of pseudotyped viruses were evaluated using Huh 7 (Japanese Collection of Research Bioresources, 0403) cells by 3-fold serial dilutions. Chemiluminescence signals were detected 24 h after the incubation of cells and virus at 37°C with 5% CO₂. The Britelite plus reporter gene assay system (PerkinElmer, Waltham, MA) and PerkinElmer Ensight luminometer were used for signal collection. The detailed procedure was described in our previous paper [12 (link)].

SARS-CoV-2-S pseudotyped virus Omicron BA.2.12.1 (DR-XG-C015), Omicron BA.4 & BA.5 (DR-XG-C013), Omicron BF.7 (DR-XG-C020), Omicron BQ.1.1 (DR-XG-C021), Omicron XBB (DR-XG-C022) and XBB.1.5 (DR-XG-C031) were purchased from Guangzhou DARUI Biotechnology Co., Ltd., and the VSV-based pseudotyped SARS-CoV-2 variants were produced by transfecting 293 T cells with S protein expression plasmids and simultaneously infected with G*ΔG-VSV (Kerafast, Boston, MA). 2 × 10⁴ Huh-7 cells were seeded in a 96-well plate. The antibodies at different dilution concentrations were mixed with SARS-CoV-2 (650 TCID₅₀/well) and pretreated for 1 hour at 37 °C, and then 200 μL mixtures were inoculated onto a monolayer of Huh-7 cells. Chemiluminescence signals were detected twenty-four hours after the incubation of cells and virus at 37 °C with 5% CO₂. The Britelite plus reporter gene assay system (PerkinElmer, Waltham, MA) and PerkinElmer Ensight luminometer were used for signal collection. The inhibition of antibodies and the value of NT₅₀ were calculated from luciferase expression of pseudotyped SARS-CoV-2. Two independent experiments were performed with triplicate or octuplicate infections, and one representative is shown.

After quantification by RT-PCR, the pseudotyped virus was diluted to the same particle number, and 100 μL aliquots were added into 96-well cell culture plates. Cells of the assayed cell lines were then digested with trypsin and added into each well at 2 × 10⁴/100 μl. Chemiluminescence monitoring was carried out after a 24 h incubation with 5% CO₂ at 37°C. The supernatant was adjusted to 100 μL for each sample. Luciferase substrate was mixed with cell lysis buffer (Perkinelmer, Fremont, CA) and was added to the plate (100 μl/well). Two minutes later, 150 μL of lysate was transferred to opaque 96-well plates. The luminescence signal was detected using a PerkinElmer Ensight luminometer, with data collected in terms of relative luminescence unit (RLU) values. Each group contained two replicates, and these experiments were repeated three times.