Diagnostic performance and interchangeability of two commercial anti-RSV-IgG and IgA ELISAs across clinically...

Accurate assessment of anti-respiratory syncytial virus (RSV)-specific antibodies has gained importance following the introduction of RSV vaccines and long-acting monoclonal antibody prophylaxis. However, the analytical performance and interchangeability of commercially available RSV-ELISAs remain insufficiently defined, leaving uncertainty about their reliability for clinical diagnostic practice. We performed a head-to-head comparison of the widely used commercial Euroimmun- and Virotech‒ELISA for detection of total anti-RSV‒IgG and IgA in 462 sera from four cohorts: healthy adults (n = 100), pregnant women (n = 59), children (n = 101), and vaccinated transplant recipients (n = 101; baseline and day + 30). Prefusion-F (pre-F)-specific IgG was quantified to infer functionally relevant antibody responses. Qualitative agreement, quantitative bias, correlations, and age-dependent patterns were assessed. Qualitative concordance in anti-RSV‒IgG detection was high (83.8%) between assays. However, the Virotech‒ELISA systematically yielded higher IgG detection rates and quantitative values (P < 0.001). Total anti-RSV‒IgG correlated significantly with pre-F‒IgG in all cohorts, with strongest correlations in children (r_s = 0.92) and vaccinated transplant recipients (r_s = 0.79). Anti-RSV‒IgA was detected infrequently (6%-11%), with moderate (79.4%) inter-assay concordance. IgG‒IgA correlations were weak in healthy adults and pregnant women (r_s < 0.25), but moderate in children and vaccinated transplant recipients (r_s ≥ 0.40). The Euroimmun- and Virotech‒ELISAs are not analytically interchangeable and exhibit systematic assay-dependent differences. Integrated analysis of total anti-RSV‒IgG, pre-F‒IgG, and IgA across clinically relevant populations emphasizes the need for assay- and population-aware interpretation to support reliable use of RSV serology in seroepidemiological studies and diagnostic laboratory practice.IMPORTANCEWith the recent introduction of espiratory syncytial virus (RSV) vaccines and infant monoclonal antibody prophylaxis, serological testing is gaining relevance in vaccine immunogenicity trials, post-licensure evaluations, and population-based serosurveillance. Although commercial ELISAs offer the potential to harmonize antibody testing, their appropriate application requires a detailed understanding of assay-specific performance and calibration characteristics. Inadequate validation can limit inter-laboratory comparability, such that identical samples yield different quantitative results across centers, thereby complicating longitudinal antibody monitoring and harmonized analysis in multicenter surveillance and vaccine studies. In this context, the present study provides a systematic cross-comparison of two widely used RSV-ELISAs and demonstrates that, despite good qualitative agreement, they are not quantitatively interchangeable and exhibit systematic differences in IgG and IgA measurements across populations and immune contexts. By characterizing the performance of total IgG, pre-F-specific IgG, and IgA in diverse clinical settings, this work supports informed assay selection and interpretation of RSV serology in clinical diagnostic practice.