Comparative assessment of four virus neutralization assays for detection of SARS-CoV-2 neutralizing antibodies

Neutralizing antibodies (NAbs) targeting receptor-binding domain (RBD) or spike of SARS-CoV-2 play an important role in blocking virus entry to the host cells and detecting their levels is critical for the assessment of humoral protective immune response following vaccination or recovery from SARS-CoV-2 infection. Here, we compared the performance of four virus neutralization tests to measure neutralizing antibodies in various sample types. We analyzed 25 serum samples obtained from mice or rabbits immunized with different vaccine platforms, and also 11 mouse anti-RBD monoclonal antibodies (MAbs) using surrogate virus neutralization test (SVNT), pseudovirus neutralization test (PVNT), conventional virus neutralization test (CVNT), and one-step or two-step inhibition flowcytometry virus neutralization test (IFVNT). All four VNTs showed significant correlations with each other, though PVNT and CVNT displayed significantly lower limit of detection (LoD) compared to the other two assays. In conclusion, our findings indicate that all four VNT assays give valid and accurate results and could be employed to determine the level of SARS-CoV-2 neutralizing monoclonal and polyclonal antibodies.