A tissue culture infectious dose-derived protocol for testing of SARS-CoV-2 neutralization of serum antibodies on adherent cells

Abstract

For a cytopathic virus such as severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the neutralization capacity of serum from convalescent or vaccinated persons or of therapeutic antibodies can be tested on adherent cell cultures. Here, a simple and tissue culture infectious dose-derived protocol for assessment of neutralization of SARS-CoV-2 is described. Compared with the often applied plaque-forming unit assay, the working load is lower, and fewer manipulations of the infected cultures are required. Hence, the method is safer for the personnel.

Keywords: Cell culture; Cell-based Assays; Immunology; Microbiology.

Graphical abstract

Figure 1

Preparation of 96-well plate for serum dilution and virus neutralization Serum samples in tubes are added to 96-well plates with culture medium (DMEM #2). Two-fold serial dilutions thereof are made using a multichannel pipette, so that samples ranges from a 10-fold to a 1280-fold dilution prior to addition of virus. As the volumes of serum solution and virus solution are the same, there is an additional two-fold dilution of the serum samples upon adding virus (new dilution: 20-fold–2560-fold).

Figure 2

In-process visual control of infections and neutralization After 3 days of incubation of Vero cells with SASR-COV-2 virus, visual control of cultures will enable to distinguish infection from non-infections (or successful neutralization). (A) Side view of four plates prior to inactivation of virus with formaldehyde. The upper two plates show wells with healthy cells (no infection) as indicated by the red color of the culture, while the lower two plates show infected cells as indicated by the yellow color of the cultures. (B) Culture after inactivation with formaldehyde (step 10). Wells with infected cells are more yellow. Wells with non-infected cells are redder. (C) The discharged culture wash (step 11) of plates with healthy cells is clear (left tube), as cells maintain adhered on the plate. The discharged culture wash of infected cells is more turbid (right tube) due to detachment of cell from plate. (D) The same plate as in B after staining (steps 12–15). Note that yellowish wells in B show non-confluence cell layers (cytotoxicity) in D, while the reddish well is B show confluent cell layers in D.

Figure 3

Example of stained plate with visible cytopathic effect Columns 1–10 depict samples from vaccinated persons, column 11 the positive control (cells, virus, no serum), and column 12 the negative control (cells, no virus, no serum). The serum samples are two-fold diluted, with the highest serum concentration in the bottom rows. The arrows for samples # 1, 5, 6, and 9 indicate that the neutralization titers were below detection limit (lowest serum dilution). The white hatched circles indicate the neutralization titer of the different sample.

Figure 4

Illustrated analysis of virus neutralization titer (A) The plate shows a thought neutralization assay with serum samples 2-fold diluted from 1/20 (bottom row) to 1/2560 (upper row). The first three columns from left are controls, and the last 9 columns are replicates of a samples (or different samples). The dark blue wells of the plate show intact cell cultures (confluent), while the light blue wells illustrate detached cultures, hence, infected cells. The last dark blue well before a light blue well appears, is defined as the neutralization titer. (B) Graph shows the scatter plot of the nice serum samples in A and the geometric mean and 95% confidential intervals of the samples as calculated in Graph Pad Prism version 8.0.

Figure 5

Example of quantification of virus neutralization in serum samples from vaccinated, non-vaccinated (naïve) and from convalescent individuals Thirty four serum samples from persons that had been vaccinated against Covid-19, recovered from a confirmed Covid-19 disease, or from healthy and non-vaccinated individuals were tested for neutralization against SARS-CoV-2. The data are analyzed by Kruskal-Wallis test for non-parametric data and multiple comparisons were corrected using the Dunn’s test (from Graph Pad Prism 8.0). Geometric means with geometric standard deviations are shown.

Figure 6

The quality of virus neutralization does not depend upon heat-inactivation of serum Serum samples from four convalescent persons split in two aliquots each. One aliquot was heat treated at 56°C for 30 min to inactivate complement, and the other aliquot was left untreated. (A) The samples were then tested for SARS-CoV-2 neutralization in triplicates. The data were statistically analyzed by 2-way ANOVA using heat treatment (yes or no) and serum sample source (person) as factors. The p-value refers to the effect of the heat treatment. Geometric means with geometric standard deviations are shown. (B) Spearmen’s correlation plot and statistical analysis (two tailed) of heat treated versus non-treated serum samples.