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Development of a Duplex Serological Multiplex Assay for the Simultaneous Detection of Epstein-Barr Virus IgA and IgG Antibodies in Nasopharyngeal Carcinoma Patients

SIMPLE SUMMARY: IgA and IgG antibodies against Epstein-Barr virus (EBV) proteins in human serum are well-known markers for EBV-positive nasopharyngeal carcinoma (NPC). Bead-based multiplex serology assays can characterize antibodies against several antigens simultaneously; however, these assays were...

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Autores principales: Schieber, Jennifer, Pring, Miranda, Ness, Andy, Liu, Zhiwei, Hsu, Wan-Lun, Brenner, Nicole, Butt, Julia, Waterboer, Tim, Simon, Julia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10177259/
https://www.ncbi.nlm.nih.gov/pubmed/37174042
http://dx.doi.org/10.3390/cancers15092578
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author Schieber, Jennifer
Pring, Miranda
Ness, Andy
Liu, Zhiwei
Hsu, Wan-Lun
Brenner, Nicole
Butt, Julia
Waterboer, Tim
Simon, Julia
author_facet Schieber, Jennifer
Pring, Miranda
Ness, Andy
Liu, Zhiwei
Hsu, Wan-Lun
Brenner, Nicole
Butt, Julia
Waterboer, Tim
Simon, Julia
author_sort Schieber, Jennifer
collection PubMed
description SIMPLE SUMMARY: IgA and IgG antibodies against Epstein-Barr virus (EBV) proteins in human serum are well-known markers for EBV-positive nasopharyngeal carcinoma (NPC). Bead-based multiplex serology assays can characterize antibodies against several antigens simultaneously; however, these assays were, so far, specific for single antibody isotypes. Here, we describe the development of a duplex serological multiplex assay for the simultaneous detection of EBV IgA and IgG antibodies in a combined assay. The novel duplex assay decreases costs and effort for future epidemiological studies in EBV and NPC research and could serve as a model for other duplex multiplex applications. ABSTRACT: 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 ≥90%. 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 > 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.
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spelling pubmed-101772592023-05-13 Development of a Duplex Serological Multiplex Assay for the Simultaneous Detection of Epstein-Barr Virus IgA and IgG Antibodies in Nasopharyngeal Carcinoma Patients Schieber, Jennifer Pring, Miranda Ness, Andy Liu, Zhiwei Hsu, Wan-Lun Brenner, Nicole Butt, Julia Waterboer, Tim Simon, Julia Cancers (Basel) Article SIMPLE SUMMARY: IgA and IgG antibodies against Epstein-Barr virus (EBV) proteins in human serum are well-known markers for EBV-positive nasopharyngeal carcinoma (NPC). Bead-based multiplex serology assays can characterize antibodies against several antigens simultaneously; however, these assays were, so far, specific for single antibody isotypes. Here, we describe the development of a duplex serological multiplex assay for the simultaneous detection of EBV IgA and IgG antibodies in a combined assay. The novel duplex assay decreases costs and effort for future epidemiological studies in EBV and NPC research and could serve as a model for other duplex multiplex applications. ABSTRACT: 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 ≥90%. 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 > 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. MDPI 2023-04-30 /pmc/articles/PMC10177259/ /pubmed/37174042 http://dx.doi.org/10.3390/cancers15092578 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Schieber, Jennifer
Pring, Miranda
Ness, Andy
Liu, Zhiwei
Hsu, Wan-Lun
Brenner, Nicole
Butt, Julia
Waterboer, Tim
Simon, Julia
Development of a Duplex Serological Multiplex Assay for the Simultaneous Detection of Epstein-Barr Virus IgA and IgG Antibodies in Nasopharyngeal Carcinoma Patients
title Development of a Duplex Serological Multiplex Assay for the Simultaneous Detection of Epstein-Barr Virus IgA and IgG Antibodies in Nasopharyngeal Carcinoma Patients
title_full Development of a Duplex Serological Multiplex Assay for the Simultaneous Detection of Epstein-Barr Virus IgA and IgG Antibodies in Nasopharyngeal Carcinoma Patients
title_fullStr Development of a Duplex Serological Multiplex Assay for the Simultaneous Detection of Epstein-Barr Virus IgA and IgG Antibodies in Nasopharyngeal Carcinoma Patients
title_full_unstemmed Development of a Duplex Serological Multiplex Assay for the Simultaneous Detection of Epstein-Barr Virus IgA and IgG Antibodies in Nasopharyngeal Carcinoma Patients
title_short Development of a Duplex Serological Multiplex Assay for the Simultaneous Detection of Epstein-Barr Virus IgA and IgG Antibodies in Nasopharyngeal Carcinoma Patients
title_sort development of a duplex serological multiplex assay for the simultaneous detection of epstein-barr virus iga and igg antibodies in nasopharyngeal carcinoma patients
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10177259/
https://www.ncbi.nlm.nih.gov/pubmed/37174042
http://dx.doi.org/10.3390/cancers15092578
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