A novel multiplex approach for the comprehensive analysis of the Epstein-Barr virus-specific humoral immune response

Background The Epstein-Barr virus (EBV) is an oncogenic herpesvirus that establishes lifelong infections in more than 90% of the human population. EBV infection is typically diagnosed using serological assays, which also distinguish between acute and persistent infection. However, current diagnostic approaches rely on a limited set of viral proteins and may therefore fail to detect disease-specific antibody signatures associated with EBV-related cancers or autoimmune diseases. Comprehensive profiling of humoral responses against the complete EBV proteome could improve the sensitivity and specificity of serological diagnostics. Methods We developed a serological multiplex assay covering all EBV proteins expressed in human cells, enabling measurement of virus-specific IgM, IgG, and IgA responses. The assay was evaluated using sera from healthy EBV carriers and EBV-negative controls. Near-infrared detection was employed to support quantitative analysis across a broad dynamic range. Performance was benchmarked against commercially available serological assays. Results The multiplex assay demonstrated a broad dynamic range with linear quantification across several orders of magnitude. Comparative evaluation showed that the assay achieved sensitivity and specificity comparable to established commercial tests while enabling comprehensive antibody profiling against the full EBV proteome. Conclusion This multiplex serological platform provides a robust approach for comprehensive characterization of EBV-specific humoral immune responses. By extending antibody profiling beyond the limited antigen panels used in routine diagnostics, the assay may facilitate the identification of novel biomarkers for EBV-associated cancers and autoimmune diseases. Future applications could contribute to improved diagnostic accuracy and support the development of targeted therapeutic strategies.