SARS-CoV-2 Neutralization Assay Protocol

We used the plaque reduction neutralization test (PRNT) as a reference for this study because neutralization assays are the standard for coronavirus serologic analysis. We tested serum samples for their neutralization capacity against SARS-CoV-2 (German isolate; GISAID ID EPI_ISL 406862; European Virus Archive Global #026V-03883) by using PRNT as described with some modifications (9 (link)). We 2-fold serially diluted heat-inactivated samples in Dulbecco modified Eagle medium supplemented with NaHCO₃, HEPES buffer, penicillin, streptomycin, and 1% fetal bovine serum, starting at a dilution of 1:10 in 50 μL. We then added 50 μL of virus suspension (400 plaque-forming units) to each well and incubated at 37°C for 1 h before placing the mixtures on Vero-E6 cells. After incubation for 1 h, we washed, cells supplemented with medium, and incubated for 8 h. After incubation, we fixed the cells with 4% formaldehyde/phosphate-buffered saline (PBS) and stained the cells with polyclonal rabbit anti-SARS-CoV antibody (Sino Biological, https://www.sinobiological.com) and a secondary peroxidase-labeled goat anti-rabbit IgG (Dako, https://www.agilent.com). We developed signal by using a precipitate forming 3,3′,5,5′-tetramethylbenzidine substrate (True Blue; Kirkegaard and Perry Laboratories, https://www.seracare.com) and counted the number of infected cells per well by using an ImmunoSpot Image Analyzer (CTL Europe GmbH, https://www.immunospot.eu). The serum neutralization titer is the reciprocal of the highest dilution resulting in an infection reduction of >50% (PRNT₅₀). We considered a titer >20 to be positive.
We performed the PRNT for serum samples from Germany by using Vero E6 cells, as described (R. Wölfel et al., unpub. data, https://doi.org/10.1101/2020.03.05.20030502) (10 (link)) and 24-well plates. Before the PRNT, we heat-inactivated patient serum samples at 56°C for 30 min. For each dilution step (in duplicate), we diluted patient serum samples in 200 μL of OptiPro serum-free medium (https://www.thermofisher.com) and mixed 1:1 with 200 μL of virus solution containing 100 PFUs. We vortexed the 400-μL serum–virus solution gently, incubated at 37°C for 1 h, and then incubated each 24-well plate with 200 μL serum–virus solution. After incubation for 1 h at 37°C, we discarded supernatants, washed cells once with PBS, and supplemented them with 1.2% microcrystalline cellulose solution in Dulbecco modified Eagle medium. After 3 days, we fixed and inactivated the plates by using a 6% formaldehyde/PBS solution and stained with crystal violet.