SARS-CoV-2 Neutralizing Antibody Assay

The serum neutralizing antibody levels against SARS-CoV-2 were determined using a microneutralization test against the Washington strain and the Delta variant, as published previously [3 (link)]. Briefly, 200 TCID₅₀ of the SARS-CoV-2 test strain was incubated with two-fold dilutions of the serum samples in 96-well plates for 1 h at 37 °C. Vero 81 cells (2 × 10⁴) were then added to each well and incubated at 37 °C for 84 h. After 84 h, the cells were fixed by adding 200 µL of cold fixation solution (1:1 mixture of ethanol and methanol) to each well and incubating the plates at −20 °C for 30 min. Once the cells were fixed, the SARS-CoV-2 titer in each well was measured by quantitating the spike protein using a SARS-CoV-2-specific anti-S antibody in a standard ELISA format, and the absorbance at 405 nm was measured using a BioTek Epoch plate reader (Agilent Technologies Inc. (Wood Dale, IL, USA)). The highest serum dilution that resulted in a ≥80% reduction in absorbance when compared to the control samples (without neutralizing antibodies) was determined as the 80% microneutralization titer (MN₈₀).