Pseudotyped SARS-CoV-2 Virus Neutralization Assay

Pseudotyped SARS-CoV-2 virus pNL4-3Δenv_SARS-CoV-2-SΔ19(G614)_Ren was synthesized as previously described [13 (link),14 (link)]. Co-transfection with vector pcDNA-VSV-G was used as the control of specificity. Plasma neutralization activity was measured by pre-incubation of pNL4-3Δenv_SARS-CoV-2-SΔ19(G614)_Ren pseudovirus (10ng p24 Gag per well) with serial dilutions (1/32 to 1/8192) of decomplemented IgG-positive plasma [15 (link)]. This mixture was then incubated with Vero E6 cells for 48 h. Cells were then lysed and viral infectivity was assessed by measuring Renilla luciferase activity (Renilla Luciferase Assay, Promega, Madison, WI, USA) with luminometer Centro XS3 LB 960 (Berthold Technologies, Baden-Württemberg, Germany). The titers of neutralizing antibodies were represented as 50% inhibitory dose (ID50) using non-linear regression in GraphPad Prism Software (GraphPad, Inc., San Diego, CA, USA).