{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,10]],"date-time":"2026-01-10T01:58:06Z","timestamp":1768010286997,"version":"3.49.0"},"reference-count":29,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2023,4,30]],"date-time":"2023-04-30T00:00:00Z","timestamp":1682812800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Luminex Corp.","award":["RP-PG-0707-10034"],"award-info":[{"award-number":["RP-PG-0707-10034"]}]},{"name":"Luminex Corp.","award":["C18281\/A19169"],"award-info":[{"award-number":["C18281\/A19169"]}]},{"name":"National Cancer Institute Intramural Research Program","award":["RP-PG-0707-10034"],"award-info":[{"award-number":["RP-PG-0707-10034"]}]},{"name":"National Cancer Institute Intramural Research Program","award":["C18281\/A19169"],"award-info":[{"award-number":["C18281\/A19169"]}]},{"name":"National Institute for Health and Care Research (NIHR)","award":["RP-PG-0707-10034"],"award-info":[{"award-number":["RP-PG-0707-10034"]}]},{"name":"National Institute for Health and Care Research (NIHR)","award":["C18281\/A19169"],"award-info":[{"award-number":["C18281\/A19169"]}]},{"name":"University Hospitals Bristol","award":["RP-PG-0707-10034"],"award-info":[{"award-number":["RP-PG-0707-10034"]}]},{"name":"University Hospitals Bristol","award":["C18281\/A19169"],"award-info":[{"award-number":["C18281\/A19169"]}]},{"name":"Weston Research Capability Funding","award":["RP-PG-0707-10034"],"award-info":[{"award-number":["RP-PG-0707-10034"]}]},{"name":"Weston Research Capability Funding","award":["C18281\/A19169"],"award-info":[{"award-number":["C18281\/A19169"]}]},{"name":"NIHR Senior Investigator award to Professor Andy Ness","award":["RP-PG-0707-10034"],"award-info":[{"award-number":["RP-PG-0707-10034"]}]},{"name":"NIHR Senior Investigator award to Professor Andy Ness","award":["C18281\/A19169"],"award-info":[{"award-number":["C18281\/A19169"]}]},{"name":"Cancer Research UK Programme","award":["RP-PG-0707-10034"],"award-info":[{"award-number":["RP-PG-0707-10034"]}]},{"name":"Cancer Research UK Programme","award":["C18281\/A19169"],"award-info":[{"award-number":["C18281\/A19169"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>Epstein-Barr virus (EBV) IgA and IgG antibodies in serum from nasopharyngeal carcinoma (NPC) patients are well-established markers for EBV-positive NPC. Luminex-based multiplex serology can analyze antibodies to multiple antigens simultaneously; however, the detection of both IgA and IgG antibodies requires separate measurements. Here we describe the development and validation of a novel duplex multiplex serology assay, which can analyze IgA and IgG antibodies against several antigens simultaneously. Secondary antibody\/dye combinations, as well as serum dilution factors, were optimized, and 98 NPC cases matched to 142 controls from the Head and Neck 5000 study (HN5000) were assessed and compared to data previously generated in separate IgA and IgG multiplex assays. EBER in situ hybridization (EBER-ISH) data available for 41 tumors was used to calibrate antigen-specific cut-offs using receiver operating characteristic (ROC) analysis with a prespecified specificity of \u226590%. A directly R-Phycoerythrin-labeled IgG antibody in combination with a biotinylated IgA antibody and streptavidin-BV421 reporter conjugate was able to quantify both IgA and IgG antibodies in a duplex reaction in a 1:1000 serum dilution. The combined assessment of IgA and IgG antibodies in NPC cases and controls from the HN5000 study yielded similar sensitivities as the separate IgA and IgG multiplex assays (all &gt; 90%), and the duplex serological multiplex assay was able to unequivocally define the EBV-positive NPC cases (AUC = 1). In conclusion, the simultaneous detection of IgA and IgG antibodies provides an alternative for the separate IgA\/IgG antibody quantification and may present a promising approach for larger NPC screening studies in NPC endemic areas.<\/jats:p>","DOI":"10.3390\/cancers15092578","type":"journal-article","created":{"date-parts":[[2023,5,3]],"date-time":"2023-05-03T05:55:09Z","timestamp":1683093309000},"page":"2578","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Development of a Duplex Serological Multiplex Assay for the Simultaneous Detection of Epstein-Barr Virus IgA and IgG Antibodies in Nasopharyngeal Carcinoma Patients"],"prefix":"10.3390","volume":"15","author":[{"given":"Jennifer","family":"Schieber","sequence":"first","affiliation":[{"name":"Division of Infections and Cancer Epidemiology, German Cancer Research Center (DFKZ), 69120 Heidelberg, Germany"},{"name":"Faculty of Biosciences, Heidelberg University, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Miranda","family":"Pring","sequence":"additional","affiliation":[{"name":"Bristol Dental School, University of Bristol, Bristol BS8 1QU, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3548-9523","authenticated-orcid":false,"given":"Andy","family":"Ness","sequence":"additional","affiliation":[{"name":"Bristol Dental School, University of Bristol, Bristol BS8 1QU, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0136-651X","authenticated-orcid":false,"given":"Zhiwei","family":"Liu","sequence":"additional","affiliation":[{"name":"Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wan-Lun","family":"Hsu","sequence":"additional","affiliation":[{"name":"Data Science Center, College of Medicine, Fu Jen Catholic University, New Taipei City 242062, Taiwan"},{"name":"Master Program of Big Data in Biomedicine, College of Medicine, Fu Jen Catholic University, New Taipei City 242062, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicole","family":"Brenner","sequence":"additional","affiliation":[{"name":"Division of Infections and Cancer Epidemiology, German Cancer Research Center (DFKZ), 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Julia","family":"Butt","sequence":"additional","affiliation":[{"name":"Division of Infections and Cancer Epidemiology, German Cancer Research Center (DFKZ), 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tim","family":"Waterboer","sequence":"additional","affiliation":[{"name":"Division of Infections and Cancer Epidemiology, German Cancer Research Center (DFKZ), 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Julia","family":"Simon","sequence":"additional","affiliation":[{"name":"Division of Infections and Cancer Epidemiology, German Cancer Research Center (DFKZ), 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,4,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1765","DOI":"10.1158\/1055-9965.EPI-06-0353","article-title":"The enigmatic epidemiology of nasopharyngeal carcinoma","volume":"15","author":"Chang","year":"2006","journal-title":"Cancer Epidemiol. Biomark. Prev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"E359","DOI":"10.1002\/ijc.29210","article-title":"Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012","volume":"136","author":"Ferlay","year":"2015","journal-title":"Int. J. Cancer"},{"key":"ref_3","first-page":"225","article-title":"Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, other head and neck neoplasms, and control groups","volume":"44","author":"Henle","year":"1970","journal-title":"J. Natl. Cancer Inst."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1877","DOI":"10.1056\/NEJMoa011610","article-title":"Serologic markers of Epstein-Barr virus infection and nasopharyngeal carcinoma in Taiwanese men","volume":"345","author":"Chien","year":"2001","journal-title":"N. Engl. J. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1186\/1750-9378-8-30","article-title":"Oncogenic human papillomavirus-associated nasopharyngeal carcinoma: An observational study of correlation with ethnicity, histological subtype and outcome in a UK population","volume":"8","author":"Robinson","year":"2013","journal-title":"Infect. Agents Cancer"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1002\/hed.23318","article-title":"Human papillomavirus and Epstein\u2013Barr virus in nasopharyngeal carcinoma in a low-incidence population","volume":"36","author":"Dogan","year":"2014","journal-title":"Head Neck"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1002\/hed.21714","article-title":"High-risk human papillomavirus in nasopharyngeal carcinoma","volume":"34","author":"Singhi","year":"2012","journal-title":"Head Neck"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1002\/hed.25450","article-title":"Epstein-Barr virus and human papillomaviruses as favorable prognostic factors in nasopharyngeal carcinoma: A nationwide study in Finland","volume":"41","author":"Ruuskanen","year":"2019","journal-title":"Head Neck"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"602","DOI":"10.1016\/0046-8177(94)90227-5","article-title":"Epstein-Barr-encoded RNA in situ hybridization: Diagnostic applications","volume":"25","author":"Ambinder","year":"1994","journal-title":"Hum. Pathol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1002\/ijc.33006","article-title":"Epstein-Barr virus and human papillomavirus serum antibodies define the viral status of nasopharyngeal carcinoma in a low endemic country","volume":"147","author":"Simon","year":"2020","journal-title":"Int. J. Cancer"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1002\/ijc.2910170102","article-title":"Epstein-barr virus-specific IgA serum antibodies as an outstanding feature of nasopharyngeal carcinoma","volume":"17","author":"Henle","year":"1976","journal-title":"Int. J. Cancer"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1093\/oxfordjournals.aje.a112470","article-title":"Antibodies to epstein-barr virus capsid antigen and early antigen in nasopharyngeal carcinoma and comparison groups","volume":"106","author":"Lin","year":"1977","journal-title":"Am. J. Epidemiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1002\/ijc.2910280308","article-title":"Association of Epstein-Barr virus with nasopharyngeal carcinoma in alaskan native patients: Serum antibodies and tissue EBNA and DNA","volume":"28","author":"Lanier","year":"1981","journal-title":"Int. J. Cancer"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"406","DOI":"10.1002\/ijc.26380","article-title":"Establishment of VCA and EBNA1 IgA-based combination by enzyme-linked immunosorbent assay as preferred screening method for nasopharyngeal carcinoma: A two-stage design with a preliminary performance study and a mass screening in southern China","volume":"131","author":"Liu","year":"2012","journal-title":"Int. J. Cancer"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Yang, Y., and Cai, Y. (2021). Screening of Nasopharyngeal Carcinoma Pharynx\u2014Diagnosis and Treatmented, IntechOpen.","DOI":"10.5772\/intechopen.97398"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1305","DOI":"10.1158\/1078-0432.CCR-17-1929","article-title":"Identification of a Novel, EBV-Based Antibody Risk Stratification Signature for Early Detection of Nasopharyngeal Carcinoma in Taiwan","volume":"24","author":"Coghill","year":"2018","journal-title":"Clin. Cancer Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1845","DOI":"10.1373\/clinchem.2005.052381","article-title":"Multiplex human papillomavirus serology based on in situ-purified glutathione s-transferase fusion proteins","volume":"51","author":"Waterboer","year":"2005","journal-title":"Clin. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e00077-20","DOI":"10.1128\/JCM.00077-20","article-title":"Validation of an Epstein-Barr Virus Antibody Risk Stratification Signature for Nasopharyngeal Carcinoma by Use of Multiplex Serology","volume":"58","author":"Simon","year":"2020","journal-title":"J. Clin. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e01107-19","DOI":"10.1128\/JCM.01107-19","article-title":"Multilaboratory Assessment of Epstein-Barr Virus Serologic Assays: The Case for Standardization","volume":"57","author":"Liu","year":"2019","journal-title":"J. Clin. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"804","DOI":"10.1111\/coa.12548","article-title":"Recruitment, response rates and characteristics of 5511 people enrolled in a prospective clinical cohort study: Head and neck 5000","volume":"41","author":"Ness","year":"2016","journal-title":"Clin. Otolaryngol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Brenner, N., Mentzer, A.J., Butt, J., Michel, A., Prager, K., Brozy, J., Wei\u00dfbrich, B., Aiello, A.E., Meier, H.C.S., and Breuer, J. (2018). Validation of Multiplex Serology detecting human herpesviruses 1-5. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0209379"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1016\/j.jim.2005.11.008","article-title":"Suppression of non-specific binding in serological Luminex assays","volume":"309","author":"Waterboer","year":"2006","journal-title":"J. Immunol. Methods"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"520","DOI":"10.3389\/fimmu.2014.00520","article-title":"IgG subclasses and allotypes: From structure to effector functions","volume":"5","author":"Vidarsson","year":"2014","journal-title":"Front. Immunol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1038\/mi.2007.6","article-title":"The immune geography of IgA induction and function","volume":"1","author":"Macpherson","year":"2008","journal-title":"Mucosal Immunol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e0250721","DOI":"10.1128\/spectrum.02507-21","article-title":"Simultaneous Measurement of IgM and IgG Antibodies to SARS-CoV-2 Spike, RBD, and Nucleocapsid Multiplexed in a Single Assay on the xMAP INTELLIFLEX DR-SE Flow Analyzer","volume":"10","author":"Cameron","year":"2022","journal-title":"Microbiol. Spectr."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"H\u00e4ring, J., Hassenstein, M.J., Becker, M., Ortmann, J., Junker, D., Karch, A., Berger, K., Tchitchagua, T., Leschnik, O., and Harries, M. (2022). Borrelia multiplex: A bead-based multiplex assay for the simultaneous detection of Borrelia specific IgG\/IgM class antibodies. BMC Infect. Dis., 22.","DOI":"10.1186\/s12879-022-07863-9"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1093\/jnci\/djad012","article-title":"Recommendations for Epstein-Barr virus-based screening for nasopharyngeal cancer in high- and intermediate-risk regions","volume":"115","author":"Lam","year":"2023","journal-title":"J. Natl. Cancer Inst."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3613","DOI":"10.1200\/JCO.21.01785","article-title":"Absolute Risk of Oropharyngeal Cancer After an HPV16-E6 Serology Test and Potential Implications for Screening: Results From the Human Papillomavirus Cancer Cohort Consortium","volume":"40","author":"Robbins","year":"2022","journal-title":"J. Clin. Oncol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.jcv.2019.05.004","article-title":"First-void urine as a non-invasive liquid biopsy source to detect vaccine-induced human papillomavirus antibodies originating from cervicovaginal secretions","volume":"117","author":"Pattyn","year":"2019","journal-title":"J. Clin. Virol."}],"container-title":["Cancers"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6694\/15\/9\/2578\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:27:30Z","timestamp":1760124450000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6694\/15\/9\/2578"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,4,30]]},"references-count":29,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2023,5]]}},"alternative-id":["cancers15092578"],"URL":"https:\/\/doi.org\/10.3390\/cancers15092578","relation":{},"ISSN":["2072-6694"],"issn-type":[{"value":"2072-6694","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,4,30]]}}}