SARS-CoV-2 Variant Neutralization Assay

The titer of each variant’s stock was measured at the 50% tissue culture infectious dose (TCID50) in six replicas in a 96-well flat-bottom tissue-culture microtiter plate. Briefly, logarithmic dilutions of previously stocked virus in presence of 3 × 10⁴ VERO E6 cells were incubated for 72 h at 33 °C in 5% CO₂. The cells were observed under a microscope for cytopathic effect development and stained with Gram’s crystal violet solution (Merck KGaA, Darmstadt, Germany) plus 5% v/v formaldehyde 40% m/v (Carlo Erba SpA, Arese, Italy). The value of TCID50 mL⁻¹ was calculated with the Reed–Muench method [9 ]. After virus titration, 50 µL of 100 TCID50 was incubated with 50 µL of serial dilutions (1:10 to 1:640) of the subject’s sera in duplicate in a 96-well flat-bottom tissue-culture microtiter plate. After 1 h incubation at 33 °C in 5% CO₂, 3 × 10⁴ VERO E6 cells were added to each well. After 72 h incubation, wells were stained with Gram’s crystal violet solution as previously reported. The neutralizing titer demonstrated the maximum dilution with the reduction of 90% of cytopathic effect. A positive titer was equal to or greater than 1:10 [7 (link),8 (link)].