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

Abstract

Assessment of immunoglobulin A (IgA) antibody responses to various Epstein-Barr virus (EBV) antigencomplexes, usually involving multiple serological assays, is important for the early diagnosis of nasopharyngeal carcinoma (NPC). Through combination of two synthetic peptides representing immunodominant epitopes of EBNA1 and viral capsid antigen (VCA)-p18 we developed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) for the specific detection of EBV reactive IgG and IgA antibodies in NPC patients (EBV IgG/IgA ELISA). Sera were obtained from healthy donors (n ‫؍‬ 367), non-NPC head and neck cancer patients (n ‫؍‬ 43), and biopsy-proven NPC patients (n ‫؍‬ 296) of Indonesian and Chinese origin. Higher values of optical density at 450 nm for EBV IgG were observed in NPC patients compared to the healthy EBV carriers, but the large overlap limits its use for NPC diagnosis. Using either EBNA1 or VCA-p18 peptides alone IgA ELISA correctly identified 88.5% and 79.8% of Indonesian NPC patients, with specificities of 80.1% and 70.9%, whereas combined single-well coating with both peptides yielded sensitivity and specificity values of 90.1 and 85.4%, respectively. The positive and negative predictive values (PPV and NPV, respectively) for the combined EBNA1 plus VCA EBV IgA ELISA were 78.7% and 93.9%, respectively. In the Indonesia panel, the level of EBV IgA reactivity was not associated with NPC tumor size, lymph node involvement, and metastasis stage, sex, and age group. In the China panel the sensitivity/specificity values were 86.2/92.0% (EBNA1 IgA) and 84.1/90.3% (VCA-p18 IgA) for single-peptide assays and 95.1/90.6% for the combined VCA plus EBNA1 IgA ELISA, with a PPV and an NPV for the combined EBV IgA ELISA of 95.6 and 89.3%, respectively. Virtually all NPC patients had abnormal anti-EBV IgG diversity patterns as determined by immunoblot analysis. On the other hand, healthy EBV carriers with positive EBV IgA ELISA result showed normal IgG diversity patterns. By using EBV IgG immunoblot diversity as confirmation assay for EBV IgA ELISA-positive samples, the sensitivity and specificity for NPC diagnosis increased to 98% and 99.2%, respectively, in the Indonesian NPC samples. The use of these combined methods for seroepidemiological screening studies is proposed.

The prognostic value of IgA/[EBNA1+VCA-p18] on survival of nasopharyngeal cancer patients

“…Elevation of antibody responses to EBV may precede onset of clinical manifestation of NPC by 1 to 5 year (Yip et al, 1994;Ji MF et al, 2007). Combined EBV serological biomarkers could improve diagnostic value of NPC (Neel and Taylor, 1990;Fachiroh et al, 2006;Liu et al, 2012;Ai et al, 2013;Chang et al, 2013). Furthermore, dynamic fluctuation of antibody level after treatment of NPC raised the possibilities of humoral response to be used as prognostic marker (Yip et al, 1994).…”

“…Sensitivity, a specificity of IgA/[EBNA1+VCA-p18] is better than either IgA EBNA-1 or IgA VCAp-18 alone (Fachiroh et al, 2006). The application of this serodiagnostic tool to predict survival or recurrence in NPC requires further clinical evidence.…”

“…The method was described in previous publication (Fachiroh et al, 2006) by the use of EBNA1 and VCA-p18 peptides (Cyto-Barr, Zuidhorn, The Netherlands, kindly provided through KWF funding) in one ELISA well. All OD 450 values were normalized by subtracting the value for 1:100-diluted EBV-negative sera used in duplicate in each ELISA run.…”

“…However, this method is quite expensive to be applied in low income countries such as Indonesia. Fachiroh et al (2006) have developed serodiagnostic tools based on enzyme-linked immunosorbent assay (ELISA) to measure IgA antibody response to combination of EBV immunodominant epitopes in one assay to diagnose NPC.…”

“…All OD 450 values were normalized by subtracting the value for 1:100-diluted EBV-negative sera used in duplicate in each ELISA run. The receiver operating characteristic analysis was done to predict the cut-off value of IgA, giving best sensitivity and specificity to predict progression (Fachiroh et al, 2006).…”