Evaluation of a novel fully automated and ELISA- based assays for detecting anti- SARS- CoV-2 neutralizing antibodies

Abstract

Background: Rapid and accurate measurement of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)-specific neutralizing antibodies (nAbs) is paramount for monitoring immunity in infected and vaccinated subjects. The current gold standard relies on pseudovirus neutralization tests which require sophisticated skills and facilities. Alternatively, recent competitive immunoassays measuring anti-SARS-CoV-2 nAbs are proposed as a quick and commercially available surrogate virus neutralization test (sVNT). Here, we report the performance evaluation of three sVNTs, including two ELISA-based assays and a novel automated bead-based immunoassay for detecting nAbs against SARS-CoV-2. Method: The performance of three sVNTs, including GenScript cPass, Dynamiker, and Mindray NTAb was assessed in samples collected from SARS-CoV-2 infected patients (n=160), COVID-19 vaccinated individuals (n=163), and pre-pandemic controls (n=70). Samples were collected from infected patients and vaccinated individuals 2-24 weeks after symptoms onset or second dose administration. Correlation analysis with pseudovirus neutralization test (pVNT) and immunoassays detecting anti-SARS-CoV-2 binding antibodies was performed. Receiver operating characteristic (ROC) curve analysis was generated to assess the optimal threshold for detecting nAbs by each assay. Results: All three sVNTs showed an excellent performance in terms of specificity (100%) and sensitivity (100%, 97.0%, and 97.1% for GenScript, Dynamiker, and Mindray, respectively) in samples collected from vaccinated subjects. GenScript demonstrated the strongest correlation with pVNT (r=0.743, R2=0.552), followed by Mindray (r=0.718, R2=0.515) and Dynamiker (r=0.608, R2=0.369). Correlation with anti-SARS-CoV-2 binding antibodies was variable, but the strongest correlations were observed between anti-RBD IgG antibodies and Mindray (r=0.952, R2=0.907). ROC curve analyses demonstrated excellent performance for all three sVNT assays in both groups, with an AUC ranging between 0.99 and 1.0 (p <0.0001). Also, it was shown that the manufacturer’s recommended cutoff values could be modified based on the tested cohort without significantly affecting the sVNT performance.Conclusion: The sVNT provides a rapid, low-cost, and scalable alternative to conventional neutralization assays for measuring and expanding nAbs testing across a wide range of research and clinical settings. Also, it could aid in evaluating actual protective immunity at the population level and assessing vaccine effectiveness to lay a foundation for boosters’ requirements.

Follow‐Up and Comparative Assessment of SARS‐CoV‐2 IgA, IgG, Neutralizing, and Total Antibody Responses After BNT162b2 or mRNA‐1273 Heterologous Booster Vaccination

“…The WHO conversion factor for the test is 1 AU = 3.31 IU/mL, and the reference range is 10–400 AU/mL. We recently evaluated this new assay and reported that it has great specificity and sensitivity in comparison to two reference techniques [ 24 ].…”