2.7. Plaque Reduction Neutralization Test (Prnt)

PRNT assays were performed in a Biosafety Level 3 laboratory using a SARS-CoV-2 isolate, as previously described [62]. In brief, serum samples were heat-inactivated (56 °C for 30 min) and 2-fold diluted in Dulbecco modified Eagle medium (DMEM). Serum dilutions were mixed with an equal volume (1:1) of a virus solution containing approximately 25 focus-forming units (FFUs) of SARS-CoV-2 and incubated for 1 h at 37 °C in a 5% CO₂ incubator. Fifty microliters of the virus–serum mixtures were added to the confluent monolayers of Vero E6 cells, in 96-wells plates and incubated for 1 h at 37 °C, in a 5% CO₂ incubator to allow for the infection of the cells. A total of 100 µL of an overlay solution made of minimum essential medium (MEM) with 2% foetal bovine serum (FBS, Sigma, Saint Louis, MO, USA), penicillin (100 U/mL, Sigma, Saint Louis, MO, US), streptomycin (100 U/mL, Sigma, Saint Louis, MO, US) and 0.8% carboxy methyl cellulose (CMC, Sigma, Saint Louis, MO, USA) were then added to each well after inoculum removal. After 26 h of incubation, the overlay was removed, and the cells were fixed with a 4% paraformaldehyde (PFA) solution. Visualization of plaques was obtained with an immunocytochemical staining method using an anti-dsRNA mouse monoclonal antibody (J2, 1:10,000; Scicons, Sziràk, Hungary) for 1 h, followed by 1 h incubation with peroxidase-labelled goat anti-mouse antibodies (1:1000; Jackson ImmunoResearch Inc., West Grove, PA, USA) and a 7 min incubation with the True Blue (KPL, Gaithersburg, MD, USA) peroxidase substrate. FFUs were counted after acquisition of pictures on a flatbed scanner. The neutralization titre was defined as the reciprocal of the highest dilution resulting in a reduction of the control plaque count >50% (PRNT₅₀).