SARS-CoV-2 Antibody Neutralization Assay

To determine the neutralizing activity of antibodies, we used a surrogate viral neutralization test (C-Pass GenScript sVNT, Piscataway NJ, USA) [17 (link),18 (link)]. Briefly, serum or plasma samples were diluted according to manufacturer’s instructions and incubated with soluble SARS-CoV-2 receptor-binding domain (RBD-HRP) antigen for 30 min, mimicking a neutralization reaction. Following incubation, samples were added to a 96-well plate coated with human ACE-2 protein. RBD-HRP complexed with antibodies were removed in a wash step. The reaction was developed with tetramethylbenzidine (TMB) followed by a stop solution allowing the visualization of bound RBD-HRP to the ACE2. Since this is an inhibition assay, color intensity was inversely proportional to the number of neutralizing antibodies present in samples. Data were interpreted by calculating the percent of inhibition of RBD-HRP binding. Samples with neutralization activity of ≥ 30% indicated the presence of SARS CoV-2 RBD-interacting antibodies capable of blocking the RBD–ACE2 interaction, thus inhibiting viral entry into host cells. While this assay measures the blocking activity of those antibodies, this activity is referred to throughout the text as “percentage of neutralization” for consistency and clarity.