Virus neutralization assay

Here is our detailed protocol for SARS-CoV-2 neutralization assay. Please feel free to ask us if you have any other questions.

Neutralization assay of authentic SARS-CoV-2 

Original protocol used in research Article

Antibody-like proteins that capture and neutralize SARS-CoV-2 DOI: 10.1126/sciadv.abd3916

1. VeroE6/TMPRSS2 cells (National Institutes of Biomedical Innovation, Health and Nutrition, cat#JCRB1819) (1) were maintained in the growth medium (GM) of Dulbecco's modified Eagle medium (DMEM) (Sigma-Aldrich, cat#D-5796-500ML) supplemented with 10% fetal bovine serum (FBS) (HyClone), penicillin (100 U/mL) and streptomycin (100 μg/mL) (Thermo Fisher Scientific, cat#15140-122). The cells were used for the neutralization assay no longer than 20 passages.
2. The VeroE6/TMPRSS2 cells were seeded into 96-well plates at 3 x 10³ cells/well (100µL/well) and cultured overnight at 37°C.
3. A serial 4-fold (or appropriate step dilutions) dilutions (8 points) of antibodies (or serum) were prepared in GM, starting from the 32-fold dilution, at the BSL2 rooms. Briefly, the GM were first dispensed into 96-well plates (columns 2-12): 155 µL and 150 µL into the top lane (row A) and the other lanes (rows B-F), respectively. Then, 5 µL of antibodies (serum) was added into the top lanes (row A) and mixed well using different tips. Next, 50 µL of the row-A solution was added into the next row B and mixed well (4-fold dilution). These 4-fold dilutions were repeated until the bottom row H (finally 219 fold dilution).
    

4. 70 µL of the diluted antibody (or serum) solution was dispensed into new 96-well plates.

5. SARS-CoV-2 virus solution was diluted in the GM at 1,000TCID₅₀/100µL at BSL3 rooms.

6. 70 µL of the virus solution was added into the dispensed antibody solution of the 96-well plates (see step #4). Final concentrations of the antibodies (or serum) are rangingbetween 2⁶ and 2²⁰ dilution.

7. The mixtures of antibody (or serum) and virus were incubated for 60 min at 37°C.

8. Medium was removed from the VeroE6/TMPRSS2 cells just prior to adding the antibody-virus mixtures.

9. The mixtures were added onto the cells and incubated for 60 min at 37°C.

10. Infected cells were washed once with 100 µL GM and incubated with 100 µL of fresh GM (prewarmed at 37°C).

11. 48 hr after infection, the culture supernatants were collected into new 96-well plates.

12. 2.5 µL of the supernatant was assayed for quantifying SARS-CoV-2 genome RNA by RT-qPCR with PrimeDirect Probe RT- qPCR Mix (Takara Bio, cat#RR650A)

using a primer/probe set for N2 (2). The RT-qPCR was performed according to the default condition of the manufacture's protocol (Takara Bio, cat#RR650A).

13. In the left column #1, positive (rows A and B) and negative (rows C and D) controls were set in duplicate. The row E-H blocks were used for standard curve samples.

References

1. Matsuyama S, Nao N, Shirato K, Kawase M, Saito S, Takayama I, Nagata N, Sekizuka T, Katoh H, Kato F, Sakata M, Tahara M, Kutsuna S, Ohmagari N, Kuroda M, Suzuki T, Kageyama T, Takeda M. 2020. Enhancedisolation of SARS-CoV-2 by TMPRSS2-expressing cells. Proc Natl Acad Sci U S A 117:7001-7003. https://doi.org/10.1073/pnas.2002589117.
2. Shirato K, Nao N, Katano H, Takayama I, Saito S, Kato F, Katoh H, Sakata M, Nakatsu Y, Mori Y, Kageyama T, Matsuyama S, Takeda M. 2020. Development of Genetic Diagnostic Methods for Detection for Novel Coronavirus 2019(nCoV-2019) in Japan. Jpn J Infect Dis 73:304-307. https://doi.org/10.7883/yoken.JJID.2020.061.

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