2.8. Seroneutralization Assay

Twenty-five microliters of complete EMEM with 10% FBS was added to each well of a 96-well clear–flat-bottom white microplate with opaque walls (Greiner Bio-One), and 25 µL of each serum was added to the first line of wells (Figure S5). Twenty-five microliters of S pseudovirus preparation (described in Section 2.6 above) diluted in complete EMEM with 10% FBS (corresponding to ~10⁴ relative luciferase units (RLUs); ~3–5µL of the initial preparation) was added to each well and left to incubate at room temperature for 1.5 h. Final volume for each well reached 50 µL; therefore, the sera dilution was doubled, 1:4–1:8–1:16–1:32–1:64–1:128–1:256–1:512. Next, 50 µL of complete EMEM with 10% FBS, containing 10⁴ HEK/ACE2/TMPRRS2/Puro cells, was added to each well and left for 60 h at 37 °C and 5% CO₂.

Plates were read by adding 25 µL of complete EMEM containing luciferin to each well just before the reading of the microplate with the luminometer (Victor, Perkin Elmer). A negative control was established without serum, which was substituted with complete medium. The RLUs were compared and normalized to those derived from wells where pseudovirus was added in the absence of sera (100%). Neutralization titer 50 (NT50) was expressed as the maximal dilution of the sera where the reduction of the signal is ≥50%. Worthy of note, the titer had to be multiplied by 40 because the initial volume of the sera tested was 0.025 mL and it had to be normalized to 1 mL.