Two-Step Epstein-Barr Virus Immunoglobulin A Enzyme-Linked Immunosorbent Assay System for Serological Screening and Confirmation of Nasopharyngeal Carcinoma

Abstract

Undifferentiated nasopharyngeal carcinoma (NPC; WHO type III) is 100% associated with Epstein-Barr virus (EBV) infection and the fourth most prevalent cancer in Indonesian males. Therapy failure is high, since most patients come to the hospital at an advanced stage of disease. Screening for early-stage NPC is needed. Here, a simple and economical two-step enzyme-linked immunosorbent assay (ELISA) system is proposed for diagnosing NPC in high-risk populations, employing the peptide-based immunoglobulin A (IgA) EBNA1 plus viral capsid antigen p18 ELISA as an initial screening test and the IgA early antigen (EA) ELISA using a different set of EBV antigens as a confirmation test. A total of 151 NPC patients and 199 regional healthy EBV carriers were used to evaluate the two-step ELISA approach. Routinely, EBV IgG immunoblotting is used as a standard confirmation test. The sensitivity and specificity for diagnosing NPC by the two-step ELISA approach increased from 85.4% to 96.7% and 90.1% to 98%, respectively, with positive predictive values and negative predictive values increasing from 78.7 and 93.9% to 97.3 and 97.5%, respectively, relative to the immunoblotting confirmation system. On discrepant samples, additional testing was done by EBV DNA load quantification in blood. Results showed that 5/11 discrepant NPC samples with an elevated IgA EA ELISA also had elevated an EBV DNA load in the circulation (range, 3,200 to 25,820 copies/ml). Therefore, the IgA EA ELISA is proposed as a confirmation test in first-line NPC serological screening studies. This two-step EBV ELISA system provides a standardized approach for NPC screening and may be used in combination with dried blood sampling in future field studies for identification of early-stage NPC in high-risk regions.

Primary Treatment Results of Nasopharyngeal Carcinoma (NPC) in Yogyakarta, Indonesia

“…These antibody responses against defined viral antigens are the basis of a proposed screening test for NPC in high-risk populations [29]–[31]. Recent insight in the molecular basis and diversity of anti EBV IgA and IgG responses allowed the development of more defined serological tools [32]–[36]. Importantly, such initial NPC-risk analysis can be done in the regional hospital setting with small volumes of blood, collected by finger prick sampling on filter paper, thus providing a cheap approach [35], [37].…”

Epstein‐Barr virus mRNA profiles and viral DNA methylation status in nasopharyngeal brushings from nasopharyngeal carcinoma patients reflect tumor origin

“…Paired serum samples available for 63 NPC patients were analysed for IgA antibodies against EBV‐specific immunodominant epitopes of EBNA1, VCAp18 and Zebra using synthetic peptide‐based ELISA assays as detailed by us previously . The cutoff value (CoV) was defined as mean optical density (OD450) value of 20 healthy regional controls + 2× standard deviation . The IgA‐ELISA CoV for EBNA1, Zebra and VCA‐p18 was 0,17, 0,28 and 0.35 OD 450 units, respectively.…”

Performance of Two Commercially Available Automated Immunoassays for the Determination of Epstein-Barr Virus Serological Status

“…In these two situations, antibodies against viral capsid antigen (VCA) IgG and VCA IgM and EBV nuclear antigen 1 (EBNA IgG) are the EBV serological markers most frequently sought for detection, since they allow determination of serological profiles characterizing a primary infection, a past infection, or the absence of EBV infection. Additionally, detection of IgA against VCA and EBNA and against the early antigens (EA) may have some utility for the screening of NPC (30). Other EBV antigens are less frequently used for EBV serology testing (5,20).…”