---
title: "Analytical validation of a multiplexed peptide microarray for Epstein–Barr virus serological profi"
id: "frontiers-in-immunology-12-analytical-validation-of-a-multiplexed-peptide-microarray-for-multi-antigen"
canonical_url: "https://medichelpline.com/clinical-feed/frontiers-in-immunology-12-analytical-validation-of-a-multiplexed-peptide-microarray-for-multi-antigen"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "Frontiers in Immunology"
source_url: "https://www.frontiersin.org/articles/10.3389/fimmu.2026.1919983"
published_at: "2026-09-03T00:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Analytical validation of a multiplexed peptide microarray for Epstein–Barr virus serological profi
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/frontiers-in-immunology-12-analytical-validation-of-a-multiplexed-peptide-microarray-for-multi-antigen
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** Frontiers in Immunology
- **Source URL:** [Original Journal Publication](https://www.frontiersin.org/articles/10.3389/fimmu.2026.1919983)
- **Published At:** 2026-09-03T00:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- The source article title indicates an analytical validation study of a **multiplexed peptide microarray** designed for multi-antigen **Epstein–Barr virus (EBV)** serological profiling across varied clinical specimen types. - The article is listed in Frontiers in Immunology but the provided source text contains only site navigation and metadata; the manuscript body, methods, results, and conclusions were not included in the source provided. - No data were available in the provided source about assay design, peptide targets, panel composition, detection chemistry, or platform instrumentation. - The source did not report analytical validation parameters such as sensitivity, specificity, precision, accuracy, limit of detection, linearity, cross-reactivity, or stability studies. - Information on specimen types tested (for example, serum, plasma, dried blood spots, saliva) and any specimen-specific performance characteristics was not reported in the supplied content. - There were no reported clinical or diagnostic performance outcomes, comparative analyses against reference assays, or intended use claims in the provided excerpt. - The publication venue (Frontiers in Immunology) and topical area (methods and technologies in immunology / viral immunology) are apparent from the navigation content, but substantive article content is missing. - Because the source lacked the manuscript text, recommendations, limitations, and next-step research directions from the authors are not available for summary. - Users seeking detailed analytical or clinical performance data, protocols, or validation metrics will need to consult the full article at the journal website; those specifics were not present in the provided source material.
## Clinical Analysis & Structured Key Points
Frontiers | Analytical validation of a multiplexed peptide microarray for multi-antigen Epstein–Barr virus serological profiling across diverse clinical specimens ORIGINAL RESEARCH article Front. Immunol. , 03 September 2026 Sec. Vaccines and Molecular Therapeutics Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1919983 Published in Frontiers in Immunology Vaccines and Molecular Therapeutics 7 impact factor 11.3 citescore Part of a Research Topic High-throughput epitope discovery and immunoprevalence analysis Submission open 8650 views 4 articles Editor & Reviewers Edited by G C Guilherme Curty Lechuga Reviewed by Y J Yogita Jethmalani K L Khang Le Quy Outline Figures and Tables Figure 1 View in article Figure 2 View in article Figure 3 View in article Figure 4 View in article Figure 5 View in article Figure 6 View in article Table 1 Characteristics of the study cohort. View in article Table 2 Comparison between ELISA classification and peptide-array classification. View in article ORIGINAL RESEARCH article Front. Immunol. , 03 September 2026 Sec. Vaccines and Molecular Therapeutics Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1919983 Analytical validation of a multiplexed peptide microarray for multi-antigen Epstein–Barr virus serological profiling across diverse clinical specimens M P Milene Peterson 1 J G Joe G. L. Hunter 1 Z G Zoran Gatalica 2 I R Inga Rose 2 P S Phillip Stafford 1,3 C W Chris W. Diehnelt 1 * 1. Robust Diagnostics, LLC, Chandler, AZ, United States 2. Reference Medicine, Inc., Phoenix, AZ, United States 3. Arizona State University, Tempe, AZ, United States See more Article metrics View details Abstract Background: 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. Methods: 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. Results: 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. Conclusion: These findings support the platform as an analytically characterized, peptide-level EBV serology tool for translational research. 1 Introduction Epstein–Barr virus (EBV) is a γ-herpesvirus that infects more than 95% of adults worldwide and persists for life as a latent infection of B cells. While most primary infections are clinically silent, EBV is the causative agent of infectious mononucleosis (IM), a recognized cofactor in lymphoid and epithelial malignancies, and has been established as a necessary but not sufficient cause for the development of multiple sclerosis (MS), with seroconversion typically preceding clinical disease by a decade or more. EBV is also associated with additional autoimmune conditions, including systemic lupus erythematosus ( 1 – 6 ). Conventional EBV serology relies on enzyme-linked immunosorbent assays (ELISA) for a limited panel of whole-protein antigens, typically EBNA-1, viral capsid antigen (VCA), and early antigen-D (EA-D) ( 6 , 7 ). These assays effectively classify exposure and infection stage but do not resolve antibody responses across the broader EBV proteome. Consequently, questions regarding how EBV-directed humoral responses are distributed across antigens, concentrated within specific regions of individual proteins, or differ between clinical states are difficult to address using conventional formats. Because whole-protein assays aggregate antibody binding across an entire antigen into a single measurement, individuals with similar antigen-level serology may recognize different linear regions of the same protein, information that is not retained in conventional ELISA measurements ( 8 – 10 ). Multiplexed peptide microarrays provide a complementary approach by enabling simultaneous, semi-quantitative measurement of antibody reactivity against many short linear epitopes ( 8 – 14 ). For such platforms to support clinical research, two requirements must be met: first, analytical performance such as reproducibility, sensitivity, dynamic range, and the sources of technical variance must be characterized; second, the platform must behave predictably across the conditions encountered in clinical workflows. Most blood is collected as serum, EDTA plasma, and increasingly common cell-free DNA (cfDNA) blood collection tubes that incorporate formaldehyde-releasing stabilizers for nucleic acid preservation ( 15 – 20 ). Whether such stabilizers materially affect multiplexed antibody measurement is incompletely characterized and has direct implications for studies that rely on remnant cfDNA-tube specimens. In this work, we report the analytical validation of an EBV peptide microarray comprising 108 peptides spanning nine major EBV antigens. The novelty of this study lies in the comprehensive analytical characterization of a multi-antigen EBV peptide microarray under conditions relevant to clinical and translational research applications. By evaluating reproducibility, detection characteristics, technical sources of variation, ELISA concordance, and matrix-dependent effects across serum, K2 EDTA plasma, and Streck cfDNA plasma, this work establishes a framework for broader translational use of peptide-array-based EBV serology. Through systematic comparison of these specimen types, we demonstrate reproducible, high-resolution antibody profiling while preserving peptide-level resolution beyond that obtainable with traditional antigen-level assays. We further benchmark composite antigen scores against conventional EBV serology and demonstrate broad EBV humoral reactivity across clinically heterogeneous cohorts including infectious mononucleosis (IM), multiple sclerosis (MS), and non-MS donors. The disease-cohort analyses were included to evaluate whether the analytically characterized platform captures known features of EBV immunobiology and to generate hypotheses for future studies. More detailed disease-specific investigations, including MS-focused multivariable modeling and biomarker-development studies, will be reported separately. 2 Materials and methods 2.1 Study cohorts and specimen collection A total of 329 de-identified human blood specimens were used, comprising people with MS (pwMS, n=74), non-MS EBV+ controls (n=194), infectious mononucleosis (IM, n=39), colorectal cancer (CRC, n=12), and non-CRC healthy controls (HC, n=10) ( Table 1 ). Specimens were procured from multiple commercial vendors and academic biorepositories. A matrix sub-study evaluated pre-analytical effects across three collection methods: Streck cfDNA blood collection tubes (n=22; formaldehyde-releasing stabilizer), K2 EDTA plasma tubes (n=18), and serum separator tubes (n=10). The Streck sub-cohort comprised CRC patients (n=12) and non-cancer controls (n=10) and was used to assess matrix-associated effects on IgG and IgM detection. For benchmarking against established serology, paired ELISA results for EBNA-1 IgG, VCA-p18 IgG/IgM, and EA-D IgG were obtained from a SeraCare reference cohort and a Discovery Life Sciences validation cohort. Laboratory personnel were not formally blinded to clinical group assignment, which is acknowledged as a limitation; however, array processing and feature extraction were performed using standardized workflows before group-level statistical analysis. Table 1 Characteristic Non-MS MS¹ IM Non-CRC CRC Participants, n 194 74 39 10 12 Female, n 85 34 14 7 9 Male, n 69 9 5 3 3 Not identified, n 40 31 20 0 0 Age (± SD), years 42.2 ± 16.6 45.1 ± 13.2 32.2 ± 17.4 57.5 ± 5.5 61.5 ± 11.8 K2 EDTA, n 50 8 0 0 0 Plasma, n 20 66 0 0 0 Serum, n 124 0 39 0 0 Streck plasma, n 0 0 0 10 12 Characteristics of the study cohort. MS, multiple sclerosis; IM, infectious mononucleosis; CRC, colorectal cancer. ¹For the MS cohort, clinical phenotype and treatment status were not available. Specimen sources included Sonora Quest, SeraCare, Precision for Medicine, Discovery Life Sciences (DLS), Creative Testing Solutions (CTS), Arizona State University, Banner Health, Boca Biolistics, and Reference Medicine. 2.2 Peptide array design The array comprised 108 synthetic peptides representing immunodominant regions from nine EBV proteins: EBNA-1, EBNA-2, EBNA-3, EBNA-4, EBNA-6, BMRF1 (EA-D), BLRF2 (VCA-p23), BFRF3 (VCA-p18), and BLLF1 (gp350/220), covering both latent and lytic phases ( Supplementary Table 1 ) ( 21 – 23 ). Peptides were 15-mers with a 10-residue tiling overlap and a C-terminal Gly-Lys-Cys linker for oriented surface attachment ( 8 ). Peptides were synthesized by MilliporeSigma (The Woodlands, TX) and spotted in triplicate on Schott Nexterion H functionalized slides (Schott Applied Microarrays, Tempe, AZ). Positive control features included immunodominant peptides from polio and other common vaccine-derived antigens to serve as internal benchmarks of assay performance. 2.3 Binding assay and image acquisition Specimens were diluted 1:400 in incubation buffer and incubated with array slides overnight at 4 °C with continuous agitation. The 1:400 working dilution was selected from a five-point dilution series to maximize signal-to-threshold ratio while avoiding high-dose hook effects observed in EBV-reactivated donors at 1:200 ( Supplementary Figure 5 ). After PBST washes, antibody binding was detected with DyLight555-conjugated goat anti-human IgG (1:1, 000) and DyLight650-conjugated goat anti-human IgM (1:10, 000), incubated 1 h at room temperature in the dark. Slides were washed sequentially with PBST, PBS, and HPLC-grade water, dried under nitrogen, and scanned on an Agilent DNA Microarray Scanner at 550 nm (IgG) and 640 nm (IgM). Fluorescence intensities were extracted with Spotxel (SICASYS Software, Germersheim, Germany); per-feature mean, median, standard deviation, and CV were retained for downstream analysis. 2.4 Data processing and statistical analysis Analyses were performed in JMP (18.0, SAS Institute, Cary, NC) and Microsoft Excel (Microsoft, Redmond, WA). Analytical performance was assessed using two QC datasets: 54 technical replicate slides of a single control specimen (RBD-22-3014) spanning eight experiments and two laboratory locations (downtown Phoenix and the SanTan facility in Chandler, AZ), and a five-point dilution series (1:200–1:3, 200) of six donors representing EBV-acute, EBV-latent, EBV-reactivated, and EBV-seronegative states. Within-block CV, between-slide CV, detection threshold (mean + 2 SD of slide-global background), limit of detection (probit regression on the dilution series at P[detect]=0.95), and minimum detectable fold-change (MDFC; estimated two ways, an empirical, distribution-free 95th percentile of |log 2 ratio| across replicate-slide pairs, and a parametric power analysis at triplicate N = 3 replicates) were computed. Variance decomposition by linear mixed model on log 2 (MFU) evaluated laboratory location, slide-manufacturing batch, and scanner-calibration contributions, with per-feature Type II F-tests and Benjamini–Hochberg FDR correction (q 8) 20 0 0 7 EBNA1 CAS (cutoff >17) 20 0 0 7 EA-D IgG BMRF1_206-220-peptide (cutoff >7) 15 4 5 3 EA-D CAS (cutoff >8) 12 5 8 2 VCA IgG BFRF3_161-175-peptide (cutoff >8) 23 8 4 11 VCA CAS (cutoff >20) 23 13 4 6 VCA IgM BFRF3_156-170-IgM peptide (cutoff >8) 13 3 3 15 VCA-IgM CAS (cutoff >56) 14 7 2 11 Comparison between ELISA classification and peptide-array classification. ELISA positivity/negativity was assigned using manufacturer-provided interpretive criteria. Positivity thresholds for peptide-array features and composite antigen scores (CAS) were established using the SeraCare reference panel (n=21) and s
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