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

Jennifer Schieber; Miranda Pring; Andy Ness; Zhiwei Liu; Wan-Lun Hsu; Nicole Brenner; Julia Butt; Tim Waterboer; Julia Simon

doi:10.3390/cancers15092578

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

Cancers (Basel). 2023 Apr 30;15(9):2578. doi: 10.3390/cancers15092578.

Authors

Jennifer Schieber^{1

2}, Miranda Pring³, Andy Ness³, Zhiwei Liu⁴, Wan-Lun Hsu^{5

6}, Nicole Brenner¹, Julia Butt¹, Tim Waterboer¹, Julia Simon¹

Affiliations

¹ Division of Infections and Cancer Epidemiology, German Cancer Research Center (DFKZ), 69120 Heidelberg, Germany.
² Faculty of Biosciences, Heidelberg University, 69120 Heidelberg, Germany.
³ Bristol Dental School, University of Bristol, Bristol BS8 1QU, UK.
⁴ Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD 20892, USA.
⁵ Data Science Center, College of Medicine, Fu Jen Catholic University, New Taipei City 242062, Taiwan.
⁶ Master Program of Big Data in Biomedicine, College of Medicine, Fu Jen Catholic University, New Taipei City 242062, Taiwan.

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.

Keywords: EBV; IgA; IgG; Intelliflex; NPC; antibodies; multiplex serology.

Grants and funding

The IntelliFlex device for this study was temporarily provided by Luminex Corp. (Austin, TX, USA), and TW received support from Luminex Corp. for the purchase of reagents. The Taiwan study was supported by the National Cancer Institute Intramural Research Program. The Head and Neck 5000 study was a component of independent research funded by the National Institute for Health and Care Research (NIHR) under its Programme Grants for Applied Research scheme (RP-PG-0707-10034). The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. Core funding was also provided through awards from Above and Beyond, University Hospitals Bristol and Weston Research Capability Funding and the NIHR Senior Investigator award to Professor Andy Ness. Human papillomavirus (HPV) serology was supported by a Cancer Research UK Programme Grant, the Integrative Cancer Epidemiology Programme (grant number: C18281/A19169).