Development of a Rapid Focus Reduction Neutralization Test Assay for Measuring SARS-CoV-2 Neutralizing Antibodies

Abstract

SARS-CoV-2 is a recently emerged human coronavirus that has escalated to a pandemic. There are currently no approved vaccines for SARS-CoV-2, which causes severe respiratory illness or death. Defining the antibody response to SARS-CoV-2 will be essential for understanding disease progression, long-term immunity, and vaccine efficacy. Here we describe two methods for evaluating the neutralization capacity of SARS-CoV-2 antibodies. The basic protocol is a focus reduction neutralization test (FRNT), which involves immunostaining infected cells with a chromogen deposit readout. The alternate protocol is a modification of the FRNT that uses an infectious clone-derived SARS-CoV-2 virus expressing a fluorescent reporter. These protocols are adapted for use in a high-throughput setting, and are compatible with large-scale vaccine studies or clinical testing. © 2020 Wiley Periodicals LLC Basic Protocol: Focus reduction neutralization test Alternate Protocol: mNeonGreen-based focus reduction neutralization test (FRNT-mNG).

Keywords: SARS-Cov-2; antibody neutralization; high-throughput neutralization assay; neutralizing antibodies; serological assay.

Figure 1.. Overview of high-throughput FRNT assay for measuring SARS CoV-2 neutralizing antibodies.

Flow chart demonstrating the experimental outline. In brief, the specimens are serially diluted and mixed with equal parts of icSARS-CoV-2 following 1hour incubation at 37° C and 5% CO₂. Then the immune complex mixture is overlaid on top of Vero-E6 cells and incubated at 37° C and 5% CO₂ for 1 hour. The viral inoculum is removed and replaced by 0.85% methyl cellulose. Infected cultures are incubated for 24 hour at 37° C and 5% CO₂. After which, the cells are fixed with paraformaldehyde and probed for SARS-CoV-2 spike protein. Foci are visualized via chromogen deposit, recorded using CTL ImmunoSpot S6 Universal Analyzer, and quantified using Viridot.

Figure 2.. FRNT-Assay plate layout and specimen dilution.

The specimens are heat inactivated and serially diluted at threefold with serum free DMEM with a 1/20 starting dilution. Initially 9 μl of the specimen is mixed with 81 μl of DMEM (becomes 1/20 after adding the viral inoculum) a total of 90 μl in row-1 of a 96 well U-bottomed plate. The specimens are further diluted three-fold by transferring 30 μl from row-1 to row-2. This three-fold serial dilution is carried all the way to the last row of the plate. The last two columns on a plate are assigned for the controls. The controls include virus alone layered on top of Vero-E6 cells, with no specimen and mock (no virus) that does not have either virus or specimen.

Figure 3.. Visualization and quantification of a representative FRNT assay.

A) Representative FRNT assay results show a comparison between the viral foci of a convalescent patient versus a healthy control at three different time points, 24, 48 and 72 hours along with a mock infection control. Antibody neutralization is quantified by counting the number of foci for each sample using the Viridot program (Katzelnick et al., 2018). B) The percentage neutralization of a convalescent patient is shown here. Each specimen is tested in two independent assays performed at different times. The FRNT₅₀ titer is interpolated using a 4-parameter nonlinear regression in GraphPad Prism 8.4.3. C) The dot plot depicted here shows FRNT₅₀ titers of 9 healthy control individuals versus 10 convalescent patients.

Figure 4.. Visualization and quantification of a representative FRNT-mNG assay.

A) Representative FRNT-mNG assay results show a comparison between the viral foci of a convalescent patient versus a healthy control at three different time points, 24, 48 and 72 hours along with a mock infection control. Antibody neutralization is quantified by counting the number of foci for each sample using the Viridot program (Katzelnick et al., 2018). B) The percentage neutralization of a convalescent patient is shown here. Each specimen is tested in two independent assays performed at different times. The FRNT₅₀ titer is interpolated using a 4-parameter nonlinear regression in GraphPad Prism 8.4.3. C) The dot plot depicted here shows FRNT₅₀ titers of 9 healthy control individuals versus 10 convalescent patients.