Microneutralization Assay for SARS-CoV-2 Variants

SARS-CoV-2 strain G614 and VOCs (Delta and Omicron) were isolated from patients in Pavia and used for microneutralization assay [24 (link), 25 ]. Briefly, 50 μL of the sample, starting from 1:10 in a serial twofold dilution series (up to 1:640), was added to two wells of a flat-bottom tissue-culture microtiter plate (COSTAR, Corning Incorporated), mixed with an equal volume of 100 Tissue Culture Infection Dose 50 (TCID50) of a SARS-CoV-2 strain, previously titrated and incubated at 33°C in 5% CO₂. All dilutions were made in Eagle’s minimum essential medium with addition of 1% penicillin, streptomycin, and glutamine and 5 μg/mL of trypsin. After 1 h of incubation at 33 °C in 5% CO₂, VERO E6 cells (VERO C1008 (Vero 76, cloneE6, Vero E6); ATCC® CRL-1586™) were added to each well. After 48 h of incubation at 33°C in 5% CO₂, wells were stained with Gram’s crystal violet solution (Merck) plus 5% formaldehyde 40% m/v (Carlo ErbaSpA) for 30 min. Microtiter plates were then washed in running water. Wells were scored to evaluate the degree of cytopathic effect (CPE) compared with the virus control. Blue staining of wells indicated the presence of neutralizing antibodies. The neutralizing titer was defined as the maximum dilution giving a reduction of 90% of the CPE. The cut-off for positivity was ≥1:10. Positive and negative controls were included in all test runs.