Pseudovirus-neutralization assay

Codon-optimized SARS-CoV-2 S gene was inserted into the pcDNA3.1 vector to construct plasmids encoding the spike proteins of SARS-CoV-2. The 293 T cell line (ATCC, CRL-3216) was transfected with the spike protein-expressing plasmids and then infected with G*ΔG-VSV virus (Kerafast, EH1020-PM). After culturing, the pseudovirus-containing supernatant was collected, filtered, aliquoted, and frozen at −80 °C for future use. Pseudovirus-neutralization assays were conducted on the Huh-7 cell line (Japanese Collection of Research Bioresources (JCRB), 0403).

Monoclonal antibodies or plasma were serially diluted in DMEM (Hyclone, SH30243.01) and incubated with pseudovirus in 96–well plates at 5% CO₂ and 37 °C for 1 h. Digested Huh-7 cell (JCRB, 0403) were seeded and cultured for 24 h. Half of the supernatant was then discarded and Bright-Lite Luciferase Assay Substrate (lyophilized) was mixed with Bright-Lite Luciferase Assay Buffer (Vazyme, DD1209-03-AB) and then the mixture was added to react in the dark. The luminescence value was detected using a microplate spectrophotometer (PerkinElmer, HH3400). IC₅₀ was determined by a four-parameter logistic regression model using PRISM (version 9.0.1).