Luciferase assay system

Recombinant VSV-based pseudotyped 2019-nCoV was obtained from the Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, NIFDC, including Delta (B.1.617.2) variant, Omicron (B.1.1.529) variant, Omicron (BA.4/5) variant and WH-1 strain. The assays were conducted according to a previous method (21 (link)). After inactivation at 56°C for 30 min, a 3-fold serial dilution of mouse serum was mixed with 650 median tissue culture infectious dose (TCID₅₀) of pseudoviruses, followed by incubation at 37°C for 60 min. Vero cells (2 × 10⁵) were added, followed by incubation at 37°C and 5% CO₂ for 24 h. NAbs were detected by luciferase expression to determine the amount of pseudoviruses entering the target cells. A luciferase assay system (PerkinElmer, MA, USA) was used to detect the luciferase activity. We included a virus control containing both virus and cells and a negative control containing only cells. The half-maximal effective concentration was determined for each tested sample. For neutralising titre <30, the value was recorded as 30 for plotting.

Mice serum samples were inactivated in a 56°C water bath for 30 min. The assay was performed in accordance with previously described methods (23 (link)). In short, serially diluted sera were separately mixed with 650 TCID50 (50% tissue culture infectious dose) of luciferase-expressing VSV-based Wuhan-Hu-1, Delta (B. 1. 617. 2), Omicron (B. 1. 1. 529), Omicron (BA. 2), Omicron (BF. 7), Omicron (BA. 2. 75), Omicron (BA. 2. 76), and Omicron (XBB) pseudoviruses and incubated in a 37°C incubator for 1 h. Vero cells (2 × 10^5 cells) were added, and the mixture was incubated at 37°C with 5% CO₂ for 24 h. Relative luciferase activity was measured with a luciferase assay system (PerkinElmer, MA, USA). Cell control and virus control groups were established. Percent neutralization was calculated, and the half-maximal neutralization titres of the samples were calculated using the Reed–Muench method.

Recombinant VSV-based pseudotyped SARS-CoV-2 was provided by the Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, National Institutes for Food and Drug Control, including Wuhan-Hu-1 SARS-CoV-2, Delta (B.1.617.2) SARS-CoV-2, and Omicron (B.1.1.529) SARS-CoV-2. The assay was performed as previously described [22 (link)]. Briefly, mouse sera were inactivated in a water bath at 56 °C for 30 min. Three-fold serial dilutions of serum and 650 TCID₅₀ (50% tissue culture infectious dose) of pseudovirus were mixed and incubated at 37 °C for 1 h. Vero cells (2 × 10⁵) were added and incubated at 37 °C with 5% CO₂ for 24 h. The amount of pseudovirus that entered the target cells was calculated by detecting the expression of luciferase to measure NAbs. Relative luciferase activity was measured using a luciferase assay system (PerkinElmer, Waltham, MA, USA). A negative control containing only cells, and a virus control containing only virus and cells, were included in each plate. The half-maximal effective concentration was calculated for all tested samples. For neutralizing titres <30, we recorded the value as 30 for plotting.