Neutralization assay

The GenScript USA Inc., cPass SARS-CoV-2 Neutralization Antibody Detection Kit, also known as SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) kit, was performed according to the manufacturer’s instructions (GenScript USA Inc.,). Briefly, in separate tubes, a 1 : 1 ratio of sample or control to HRP conjugated to SARS-CoV-2 receptor binding domain fragment (HRP-RBD) were pre-incubated at 37 °C for 30 min. After adding the mixtures into capture plates pre-coated with human angiotensin converting enzyme 2 (hACE2), the plates were sealed with a plate sealer and incubated at 37 °C for 15 min. The plates were washed four times with wash solution, 100 µl of TMB Solution was added to each well, and the plates were incubated in the dark at 20–25 °C for 15 min. Following the addition of 50 µl of stop solution to each well, the absorbance was immediately read using a PerkinElmer Victor XV plate reader at 450 nm. This assay detects serum or plasma antibodies that neutralize the RBD-hACE2 interaction and are interpreted by the inhibition rate, calculated using the following formula: Inhibition (%) = (1 − sample optical density value/negative control optical density value) × 100. Specimens are considered positive for neutralizing antibodies against SARS-CoV-2 RBD if the percent inhibition is ≥20 % and negative (or not detectable) if the percent inhibition is <20 %.