Antibody Responses to Recombinant Epstein-Barr Virus Antigens in Nasopharyngeal Carcinoma Patients: Complementary Test of ZEBRA Protein and Early Antigens p54 and p138

Abstract

Serological tests based on the antibodies directed against the Epstein-Barr virus early antigen (EA) and viral capsid antigen (VCA), which have been recognized as tumor markers for nasopharyngeal carcinoma (NPC), are routinely used to help in the diagnosis of this malignancy. The detection of these antibodies reveals very low titers, found only in a small proportion of young compared with older NPC patients. This is a problem for the diagnosis of NPC, especially among Maghrebians, among whom young people are also affected, and emphasizes the necessity to search for more reliable markers. The present study reports results of immunoglobulin G (IgG) and IgA responses of NPC patients to recombinant EA antigens p54 (BMRF1) and p138 (BALF2), VCA complex antigens p18 (BFRF3) and p23 (BLRF2), and EBNA antigen p72 (BKRF1). Our results show that IgA-EA-p54 and -p138 (IgA-EA-p54؉138) antibodies have a diagnostic value for detection of NPC (70%), compared with IgA-VCA-p18؉23 and IgA-EBNA-p72, which have limited diagnostic value, especially in young patients. It is also noteworthy that IgA-EA-p54؉138 can detect a high percentage (64%) of NPC cases negative by immunofluorescence. These results, however, clearly show that a single test cannot achieve the objective of detecting all NPC patients, and it seems advisable to combine different tests for the diagnosis of NPC. The combination of IgG-ZEBRA with IgA-EA-p54؉138 improved the sensitivity of detection of NPC to 95% in the overall NPC population. The use of IgA-EA-p54؉138 in combination with IgG-ZEBRA will facilitate detailed studies on the pattern of antibody response, which may result in the development of useful serological markers to guide the treatment of NPC.Epstein-Barr virus (EBV) humoral immunology has played a major role in studies dealing with a relationship between this virus and nasopharyngeal carcinoma (NPC) (12,13,24). Detection of antibodies to the EBV viral capsid antigen (VCA) and EBV early antigen (EA) in sera by indirect immunofluorescence (IF) assays was one of the earliest tests developed. To date, the IF assays still serve as the "gold standard" of EBV serodiagnosis (10,11,13). These tests showed the importance of antibodies directed against some of the serologically defined EBV antigens in the diagnosis of EBV-associated diseases. They also help in the clinical management of patients with EBV-associated malignancies. Diagnostically relevant antibodies that have been identified by a number of investigators over the years are immunoglobulin G (IgG) and IgA antibodies directed against EA and VCA. The IgA-EA test, which is routinely used in many laboratories throughout the world, is one of the more specific EBV-associated NPC diagnostic tests available. Moreover, detection of anti-IgA antibodies by IF is suitable for the identification of patients with occult NPC, and the identification of populations at high risk for the development of this cancer (3,12,19,25,30,31). However, the IF assays are time-consuming, not suitable for automatic handling, and diff...

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

“…Peptides may substitute for natural or recombinant proteins as a stable, reproducible, and cheap source of antigen in ELISA. VCA-p18, the small capsid protein of 18 kDa encoded by the BFRF3 gene, is highly immunogenic in humans, and its use in ELISA is well described (3,9,30,35,44). The EBNA1 synthetic peptide used in this study derived from a unique region of EBNA1 (combining amino acids 382 to 410 and 413 to 452) not including glycine-alanine repeat sequences, which may produce nonspecific reactivity (27,32).…”

“…However, single markers may not be sufficient to identify all individual NPC patients in view of the observed diversity of antibody reactivity among individual NPC patients. Combined testing for ZEBRA and VCA-p18 IgA or ZEBRA plus EBNA1 IgA reactivity was recently proposed for more-sensitive NPC diagnosis (1,2,9). In all cases two-marker testing required testing by two separate assays.…”

“…Previously reported EBV antigens for ELISA include EBV cell extracts (11,12,46), purified native or recombinant EBV proteins (3,7,9), and synthetic peptides (16,23,44). Among the defined EBV antigens proposed as markers in ELISA are thymidine kinase (7,25), DNase (41), ribonucleotide reductase (15), ZEBRA (9,22), VCA-p18 (35,44), EBNA1 (16,21), and EAd-p47/p138 (14). However, single markers may not be sufficient to identify all individual NPC patients in view of the observed diversity of antibody reactivity among individual NPC patients.…”

“…The assessment of anti-EBNA1, anti-EA, and anti-VCA antibodies requires separate assays, each contributing to NPC diagnosis (23). Individual NPC patients may respond quite differently to EBV lytic-cycle proteins, but the small capsid protein VCA-p18, encoded by the BFRF3 reading frame, is a highly prevalent target for antibody responses, including IgG and IgA (3,9,13,23,35,44). However, high titers of IgG against EA/VCA are not specific for NPC and can be observed in other EBV-linked diseases as well (18).…”

Dried-Blood Sampling for Epstein-Barr Virus Immunoglobulin G (IgG) and IgA Serology in Nasopharyngeal Carcinoma Screening

“…The diagnosis and screening of NPC are done mostly by indirect antibody detection using cell spot slide tests, one of the earliest serology methods developed, which to date is still used as a "gold standard" (8,35). However, these slide-based assays are subjective and cumbersome, making their application in mass screening inconvenient (11,23).…”