Associated uncertainty estimation during the validation process of TCID50 and FRNT neutralization assays against SARS-CoV-2 variants, used in population surveillance research and correlates of protection.

Neutralizing antibody (nAb) assays are essential for evaluating antibody-mediated immune response against SARS-CoV-2, in the context of efficacy and potency of vaccines, as well as population-level surveillance. This study aimed to estimate the associated uncertainty during the validation of two neutralization assays-50% tissue culture infectious dose microneutralization (TCID50MN) and fluorescent focus reduction neutralization test (FRNT)-to measure activity of nAbs against SARS-CoV-2 variants of concern (VOCs) within an ISO/IEC 17025:2017 accredited laboratory. Human serum samples were tested against five SARS-CoV-2 variants (Wuhan, BA.2, BA.5, XBB.1, and JN.1) using vesicular stomatitis virus (VSV) pseudotyped viruses. The validation process was based on international guidelines for the suitability and validation of analytical methods. Both assays demonstrated high diagnostic performance to detect neutralizing activity, where FRNT yielded higher sensitivity but greater inter-analyst variability than TCID50MN. These results highlight the importance of identifying the level of impact of factors associated with obtaining a result, such as the training of analysts and the standardization of the test. This study establishes a validated framework for nAb quantification in compliance with international standards. The declaration of associated uncertainty to each method, along with sustained internal quality control, strengthens the diagnostic capacity of these assays in clinical research of correlates of protection and immunity induced by vaccination, in addition to reinforcing transparency and regulatory compliance, as critical elements in decision-making.