Pseudo-viruses neutralization assay

Pseudo-viruses and mouse serum were generated as described above. The 50 μL serial diluted mice serum were incubated with pseudo-viruses (1 × 10⁹ copies/mL) at 37°C for 1 hour. These pseudo virus-serum mixtures were added to co-culture with hACE2-293T cells. After 72 hours, the luciferase activities of hACE2-293T cells were analyzed by the Bright-Luciferase Reporter Assay System (Promega, China). Relative luminescence unit of Luc activity was detected using the ThermoFisher LUX reader (ThermoFisher, USA). All experiments were performed at least three times and expressed as means ± SEM. Half-maximal inhibitory concentrations (IC₅₀) of dilution folds were calculated using the Dose-response-inhibition-variable slope four-parameter logistic regression in GraphPad Prism 8.0.