Analytical validation of a multiplexed peptide microarray for multi-antigen Epstein–Barr virus serological profiling across diverse clinical specimens

Abstract

Epstein–Barr virus (EBV) infects more than 95% of adults and is associated with outcomes ranging from asymptomatic latency to infectious mononucleosis (IM), malignancies, and multiple sclerosis (MS). Conventional ELISA-based EBV serology is often restricted to a small number of soluble, whole-protein antigens and does not preserve information regarding the specific linear regions of antigen recognition that contribute to the overall antibody response. Multiplexed peptide microarrays address this limitation, but their use in clinical practice requires characterization of their analytical performance and behavior across the diverse conditions encountered in practical use. Here we report the analytical validation of a 108-peptide microarray spanning nine EBV proteins, applied to 329 specimens collected as serum, EDTA plasma, and Streck cell-free DNA (cfDNA) blood collection tubes. Within-block and between-slide reproducibility, detection thresholds, and minimum detectable fold-change were consistent with established peptide-array benchmarks, and composite antigen scores correlated with ELISA assays for EBNA-1, VCA-p18, and EA-D. IgG signals were stable across collection matrices, whereas IgM was selectively attenuated in Streck plasma, indicating that fixative-containing tubes should be approached cautiously when IgM is the readout of interest. When applied to IM, MS, and non-MS donors, the peptide array consistently detected broad EBV humoral reactivity. In this proof-of-concept disease-cohort analysis, the array recapitulated biologically relevant EBNA-1 C-terminal reactivity patterns previously associated with MS-related molecular mimicry, but array-wide EBV reactivity did not yield a simple MS-discriminating signature. Although EBV reactivity has been reported to distinguish MS from non-MS cohorts, only a very small set of EBV epitopes was cohort-associated in our data, indicating that platform-level seropositivity reflects EBV exposure rather than an obvious MS-specific signal. These findings support the platform as an analytically characterized, peptide-level EBV serology tool for translational research.