SARS-CoV-2 Surrogate Virus Neutralization Test


A robust serological test for neutralizing antibodies against SARS-CoV-2 is urgently needed to determine not only the infection rate, herd immunity, and expected humoral protection, but also the efficacy of the vaccine during clinical trials and after clinical trials. large-scale vaccination. The current gold standard is the conventional virus neutralization test which requires live pathogens and a biosafety level 3 laboratory. Here, we report a SARS-CoV-2 surrogate virus neutralization assay that detects total immunodominant neutralizing antibodies directed to the receptor-binding domain of the viral spike (S) protein in an isotype and species-independent manner.

Our simple and rapid test is based on antibody-mediated blocking of the interaction between the angiotensin-converting enzyme 2 (ACE2) receptor protein and the receptor-binding domain. The test, which has been validated with two cohorts of COVID-19 patients in two different countries, achieves a specificity of 99.93% and a sensitivity of 95-100% and differentiates antibody responses to various human coronaviruses. The surrogate virus neutralization test does not require biosafety level 3 containment, making it widely accessible to the general community for both research and clinical applications.


  • Cells and viruses

Human embryonic kidney (HEK293T) cells (ATCC #CRL-3216) and African green monkey kidney clone E6 (Vero-E6) cells (ATCC #CRL-1586) were maintained in Dulbecco’s modified Eagle’s medium. (DMEM) supplemented with 10% fetal bovine serum. SARS-CoV-2, isolate BetaCoV/Singapore/2/2020 (GISAID accession code EPI_ISL_406973), was used for VNT in Vero-E6 cells.

HEK293T/SARS-CoV-2 spike-expressing cells were generated by transduction of the pQCXIH-SARS-CoV-2 spike, followed by hygromycin selection at a final concentration of 100µgml−1. SARS-CoV-2 pseudotyped spike virus was generated by infecting HEK293T-SARS-CoV-2 spike cells with ΔG-Luc vesicular stomatitis virus seed virus at a multiplicity of infection of 5 and was collected 16 h after infection.


For the indirect ELISA, MaxiSORP plates (Nunc) were coated with 100 ng of each protein using 100 µM carbonate buffer and blocked with OptEIA Assay Diluent (BD). Sera from patients with COVID-19 or SARS were tested at a dilution of 1:50 and detected by goat anti-human IgG-HRP (Santa Cruz) at a dilution of 1:10,000. For the capture, ELISA plates MaxiSORP (Nunc) were coated with 10µgml−1 of anti-human IgM (SeraCare), anti-human IgG (Jackson labs) or anti-human IgA (GenScript) in bicarbonate buffer overnight at 4°C.

Wells were blocked with the diluent of BD OptEIA (BD) assay for 1h at 37°C and heat-inactivated sera were added at a dilution of 1:50 and incubated for 1h at 37°C. After extensive washing, SARS-CoV-2 HRP–RBD (GenScript) was added at 4µgml−1 and incubated for 30min at 37°C. The chromogenic reaction was quantified after the addition of TMB substrate (Invitrogen) and stop solution (KPL SeraCare). The absorbance of the samples was measured at 450 nm and the background at 570 nm.

  • cVNT and pVNT

For cVNT, 50 μl of 2-fold serially diluted serum was preincubated with 50 μl of 1000 TCID50 per ml of SARS-CoV-2 in 5% FBS in DMEM for 90 min at 37°C. Virus and serum mixtures were then added to the Vero-E6 cell monolayer for 1h at 37°C. One hour after infection, the inoculum was removed and the infected cells were washed once with 5% FBS in DMEM. Cells were then replaced with 5% FBS in DMEM and neutralization titers were determined at 4 dpi.

For pVNT, 1.5 × 106 RLU of SARS-CoV-2 spike pseudotyped virus were preincubated with two-fold serially diluted test serum to a final volume of 50 µL for 1 h at 37 °C, followed by infection of transfected HEK293T with ACE2 cells. At 18–20 h post-infection, an equal volume of ONE-Glo luciferase substrate (Promega) was added and the luminescence signal was measured using a Cytation 5 microplate reader (BioTek) with Gen5 software (Version 3.03 .14). The same dilution range of 1:20 to 1:1,280 was used to facilitate side-by-side comparison in the correlation studies of the three different VNT assays.

  • Statistic analysis

Statistical analysis was performed using GraphPad Prism 7 software. Differences between negative control and COVID-19 test sera were analyzed using an unpaired t-test. Differences between matched SARS sera in SARS-CoV-2 sVNT and SARS-CoV sVNT were analyzed using a paired t-test. Correlations between sVNT and cVNT or pVNT were analyzed using Pearson’s correlation coefficients. All data presented is derived from two independent experiments.

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