{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T07:16:43Z","timestamp":1788419803555,"version":"build-2803163510"},"reference-count":29,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Epstein\u2013Barr 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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>These findings support the platform as an analytically characterized, peptide-level EBV serology tool for translational research.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fimmu.2026.1919983","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T06:43:36Z","timestamp":1788417816000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Analytical validation of a multiplexed peptide microarray for multi-antigen Epstein\u2013Barr virus serological profiling across diverse clinical specimens"],"prefix":"10.3389","volume":"17","author":[{"given":"Milene","family":"Peterson","sequence":"first","affiliation":[{"name":"Robust Diagnostics, LLC","place":["Chandler, AZ, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joe G. L.","family":"Hunter","sequence":"additional","affiliation":[{"name":"Robust Diagnostics, LLC","place":["Chandler, AZ, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zoran","family":"Gatalica","sequence":"additional","affiliation":[{"name":"Reference Medicine, Inc.","place":["Phoenix, AZ, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Inga","family":"Rose","sequence":"additional","affiliation":[{"name":"Reference Medicine, Inc.","place":["Phoenix, AZ, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Phillip","family":"Stafford","sequence":"additional","affiliation":[{"name":"Robust Diagnostics, LLC","place":["Chandler, AZ, United States"]},{"name":"Arizona State University","place":["Tempe, AZ, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chris W.","family":"Diehnelt","sequence":"additional","affiliation":[{"name":"Robust Diagnostics, LLC","place":["Chandler, AZ, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,3]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2022.08.026","article-title":"Epstein-Barr virus: Biology and clinical disease","volume":"185","author":"Damania","year":"2022","journal-title":"Cell"},{"key":"B2","doi-asserted-by":"publisher","first-page":"296","DOI":"10.1126\/science.abj8222","article-title":"Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis","volume":"375","author":"Bjornevik","year":"2022","journal-title":"Science"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1038\/s41582-023-00775-5","article-title":"Epstein-Barr virus as a leading cause of multiple sclerosis: mechanisms and implications","volume":"19","author":"Bjornevik","year":"2023","journal-title":"Nat Rev Neurol"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2020.623944","article-title":"Epstein barr virus and autoimmune responses in systemic lupus erythematosus","volume":"11","author":"Jog","year":"2020","journal-title":"Front Immunol"},{"key":"B5","doi-asserted-by":"publisher","first-page":"31","DOI":"10.1007\/s00432-021-03824-y","article-title":"Estimating the global burden of Epstein-Barr virus-related cancers","volume":"148","author":"Wong","year":"2022","journal-title":"J Cancer Res Clin Oncol"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1128\/jcm.00164-09","article-title":"Evidence-based approach for interpretation of Epstein-Barr virus serological patterns","volume":"47","author":"Klutts","year":"2009","journal-title":"J Clin Microbiol"},{"key":"B7","unstructured":"Laboratory testing for epstein-barr virus (EBV)\n          \n          \n          \n          2024"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.4c02806","article-title":"A peptide microarray platform approach for discovery of immunodominant antibody epitopes","volume":"96","author":"Stafford","year":"2024","journal-title":"Anal Chem"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1038\/nm.2913","article-title":"On silico peptide microarrays for high-resolution mapping of antibody epitopes and diverse protein-protein interactions","volume":"18","author":"Price","year":"2012","journal-title":"Nat Med"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1074\/mcp.m115.051664","article-title":"High-density peptide microarray analysis of IgG autoantibody reactivities in serum and cerebrospinal fluid of multiple sclerosis patients","volume":"15","author":"Hecker","year":"2016","journal-title":"Mol Cell Proteomics"},{"key":"B11","doi-asserted-by":"publisher","first-page":"45","DOI":"10.1016\/j.tibtech.2012.10.012","article-title":"Immunosignaturing: a critical review","volume":"31","author":"Sykes","year":"2013","journal-title":"Trends Biotechnol"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1074\/mcp.m115.054601","article-title":"General assessment of humoral activity in healthy humans","volume":"15","author":"Stafford","year":"2016","journal-title":"Mol Cell Proteomics"},{"key":"B13","doi-asserted-by":"publisher","first-page":"17610","DOI":"10.1038\/s41598-017-17941-7","article-title":"A simple platform for the rapid development of antimicrobials","volume":"7","author":"Johnston","year":"2017","journal-title":"Sci Rep"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1039\/c7cc08242h","article-title":"Non-natural amino acid peptide microarrays to discover Ebola virus glycoprotein ligands","volume":"54","author":"Rabinowitz","year":"2018","journal-title":"Chem Commun (Camb)"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1586\/14789450.3.4.409","article-title":"Effects of preanalytical variables on peptide and protein measurements in human serum and plasma: implications for clinical proteomics","volume":"3","author":"Rai","year":"2006","journal-title":"Expert Rev Proteomics"},{"key":"B16","doi-asserted-by":"publisher","first-page":"4","DOI":"10.1016\/j.clinbiochem.2009.10.001","article-title":"Impact of blood collection devices on clinical chemistry assays","volume":"43","author":"Bowen","year":"2010","journal-title":"Clin Biochem"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1373\/clinchem.2018.298323","article-title":"Guidelines for the preanalytical conditions for analyzing circulating cell-free DNA","volume":"65","author":"Meddeb","year":"2019","journal-title":"Clin Chem"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0166354","article-title":"Performance of streck cfDNA blood collection tubes for liquid biopsy testing","volume":"11","author":"Medina Diaz","year":"2016","journal-title":"PloS One"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1016\/j.clinbiochem.2016.03.012","article-title":"Comparative analysis of circulating tumor DNA stability In K","volume":"49","author":"Kang","year":"2016","journal-title":"Clin Biochem"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1038\/nrc.2017.7","article-title":"Liquid biopsies come of age: towards implementation of circulating tumour DNA","volume":"17","author":"Wan","year":"2017","journal-title":"Nat Rev Cancer"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0179124","article-title":"Serological profiling of the EBV immune response in Chronic Fatigue Syndrome using a peptide microarray","volume":"12","author":"Loebel","year":"2017","journal-title":"PloS One"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1001\/jamaneurol.2024.0272","article-title":"Serologic response to the epstein-barr virus peptidome and the risk for multiple sclerosis","volume":"81","author":"Cortese","year":"2024","journal-title":"JAMA Neurol"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2024.1487523","article-title":"In-depth analysis of serum antibodies against Epstein-Barr virus lifecycle proteins, and EBNA1, ANO2, GlialCAM and CRYAB peptides in patients with multiple sclerosis","volume":"15","author":"Vasilenko","year":"2024","journal-title":"Front Immunol"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1002\/eji.202049101","article-title":"Peptide microarray-based analysis of antibody responses to SARS-CoV-2 identifies unique epitopes with potential for diagnostic test development","volume":"51","author":"Holenya","year":"2021","journal-title":"Eur J Immunol"},{"key":"B25","volume-title":"SCHOTT NEXTERION\u00ae Slide H Protocol Document","year":"2024"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1128\/jcm.44.4.1459-1467.2006","article-title":"Single-assay combination of Epstein-Barr Virus (EBV) EBNA1- and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening","volume":"44","author":"Fachiroh","year":"2006","journal-title":"J Clin Microbiol"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-022-04432-7","article-title":"Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM","volume":"603","author":"Lanz","year":"2022","journal-title":"Nature"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2424986122","article-title":"Antibody reactivity against EBNA1 and GlialCAM differentiates multiple sclerosis patients from healthy controls","volume":"122","author":"Sattarnezhad","year":"2025","journal-title":"Proc Natl Acad Sci USA"},{"key":"B29","doi-asserted-by":"publisher","first-page":"e184481","DOI":"10.1172\/JCI184481","article-title":"Accumulation of Epstein-Barr virus-induced cross-reactive immune responses is associated with multiple sclerosis","volume":"134","author":"Vietzen","year":"2024","journal-title":"J Clin Invest"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2026.1919983\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T06:43:43Z","timestamp":1788417823000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2026.1919983\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,3]]},"references-count":29,"alternative-id":["10.3389\/fimmu.2026.1919983"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2026.1919983","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,3]]},"article-number":"1919983"}}