As of 2023, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate with continued emergence of variants (such as Alpha, Beta, Gamma, Delta, and Omicron) (Fiolet et al., 2022; Liu and Rocklov, 2022). Vaccines against the virus have been developed to prevent coronavirus disease 2019 (COVID-19) (Firouzabadi et al., 2023; Kherabi et al., 2022). To establish effectiveness of these vaccines, especially in light of recent variants with an increased potential of immune escape (Carabelli et al., 2023], it needs to be confirmed that methods assessing correlates of protection (CoPs) sustain accuracy and validity.

Most current COVID-19 vaccines target the SARS-CoV-2 spike (S) protein on the viral surface (Firouzabadi et al., 2023). Neutralizing antibodies that target the S protein are a known CoP for vaccine efficacy for COVID vaccines (Feng et al., 2021, Fong et al., 2023; Gilbert et al., 2022; Misra and Theel, 2022; Van Tilbeurgh et al., 2021). Microneutralization assays (MN50) are used to quantify functional neutralizing antibodies against SARS-CoV-2 in human serum (Bewley et al., 2021, Keech et al., 2020). Such neutralizing antibodies may develop as a result of natural infection or after receipt of a SARS-CoV-2 S protein vaccine (Morales-Nunez et al., 2021).

This article describes the validation of an in vitro MN50 assay (360biolabs, Melbourne, Vic, Australia) to detect/quantitate SARS-CoV-2 neutralizing antibodies against prototype and variant strains (Beta, Delta, Omicron BA.1, Omicron BA.5, and XBB.1.5). The aim was to assess suitability of the assay for detection of SARS-CoV-2 neutralizing antibodies in human serum from SARS-CoV-2 vaccine clinical trials. The microneutralization assay was assessed for precision, specificity, selectivity, linearity, robustness, stability, and other validation parameters. The MN50 assay described here is suitable for detection of SARS-CoV-2 neutralizing antibodies (a CoP for vaccine efficacy) in a robust, precise, and accurate manner, including in clinical trial serum samples.