A PRNT-anchored OD-based micro-neutralization cytopathic effect assay for rapid screening of adenovirus neutralizing...

Neutralizing antibodies (nAbs) are key indicators of protective immunity against human adenoviruses (HAdVs), but their measurement by the plaque reduction neutralization test (PRNT) is labor-intensive and poorly suited for large-scale serum screening. In this study, we describe and apply an optical density (OD)-based micro-neutralization cytopathic effect (MN-CPE) assay as a practical screening platform for adenovirus neutralizing activity. Assay conditions were optimized using HAdV-5 infection of A549 cells to achieve reproducible and complete cytopathic effects under standardized conditions. A PRNT-anchored cutoff was established using immune rabbit sera, enabling objective classification of neutralization outcomes. Application of MN-CPE to 40 human serum samples demonstrated clear separation between screening-negative and screening-positive samples, with inhibition values showing a bimodal distribution. Using a 30% inhibition threshold and a majority-rule criterion across three independent MN-CPE assay runs, 17 of 40 samples (42.5%) were classified as screening-positive for HAdV-5 neutralizing activity. Unlike reporter virus-based neutralization assays, MN-CPE uses wild-type virus and standard laboratory equipment, supporting cost-effective and accessible implementation. Although human sera were evaluated at a single dilution, the broad dynamic range of OD-derived inhibition values supported reliable discrimination of neutralizing activity under fixed assay conditions. Together, these findings support MN-CPE as an objective, OD-based, and practical screening assay for adenovirus neutralizing activity, with potential utility in large-cohort seroepidemiological studies.