SARS-CoV-2 Neutralizing Antibody Assay

A chemiluminescent assay (Liaison SARS-CoV-2 trimeric, Diasorin) was used for Spike IgG quantification. Values higher than 33.8 BAU/mL were considered positive. SARS-CoV-2 neutralizing antibody titer (NT Abs) was determined as previously reported [12 (link),13 (link)]. Briefly, 50 µL of serum in serial fourfold dilution, was placed in two wells of a flat-bottom tissue culture microtiter plate (COSTAR, Corning Incorporated, New York, NY, USA). The same volume of 100 TCID50 of SARS-CoV-2 strain was added, and the plates were incubated at 33 °C in 5% CO₂. After 1 h of incubation at 33 °C and 5% CO₂, VERO E6 cells were added to each well. After further 72 h of incubation at 33 °C and 5% CO₂, the plates were stained with Gram’s crystal violet solution (Merck KGaA, Damstadt, Germany) plus 5% formaldehyde 40% m/v (Carlo ErbaSpA, Arese, Italy) for 30 min. The microtiter plates were then washed under running water. The wells were scored to evaluate the degree of cytopathic effect (CPE) compared to the virus control. A blue staining of the wells indicated the presence of NT Abs. The neutralizing titer was the maximum dilution showing the reduction of 90% of CPE. All the experiments were performed in a BSL3 facility. Values higher or equal to 1:10 serum titer were considered positive, according to our protocol.