Neutralization assay.

Vero E6 cells were seeded in 96-well plates at a density of 1.5 × 10⁴ cells per well in complete DMEM and incubated 24 h at 37°C and 5% CO₂. SARS-CoV-2 (300 TCID50) was preincubated with serially diluted sera for 1 h at 37°C starting at sera dilution 1:8. Each sera dilution was tested in duplicate. Then, virus–sera mixture was added onto Vero E6 cells in a final volume of 100 μL in DMEM 2% FBS. After 72 h at 37°C and 5% CO₂, cultures were fixed with formaldehyde 10% at 4°C for 24 h and stained with crystal violet solution in methanol. The cytopathic effect (CPE) of the virus on the cell monolayer was assessed visually, and neutralization titer (NT) was defined as the inverse of the highest serum dilution without any CPE.