Native early antigen of Epstein–Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma

Abstract

The Epstein-Barr virus (EBV) early antigen (EA) complex consists of multiple proteins with relevance for diagnosis of acute, chronic and malignant EBV related diseases, including nasopharyngeal carcinoma (NPC). In a recent study, it was found that the molecular diversity of EBV-specific IgG and IgA antibody responses in NPC patients and demonstrated that these reflect independent B-cell triggering leading to distinct EBV antigen-recognition profiles. The fine-specificity of NPC-related IgG and IgA responses was explored further against defined recombinant and synthetic EBV-EA antigens using immunofluorescence, immunoblot and ELISA techniques and determined their diagnostic value in a large panel of sera from NPC (n = 154), non-NPC tumor patients (n = 133), acute mononucleosis patients (n = 70) and healthy EBV carriers (n = 259). Individual recombinant EBV-EA markers yielded sensitivity/specificity values not exceeding 86%, whereas selected EA-specific peptide epitopes were rather poorly recognized by IgG and IgA antibodies in NPC sera. Surprisingly, we found that a "low salt" native EA-protein extract reproducibly prepared from purified nuclei of EA-induced HH514 cells, and containing characteristic EA(D)-polypeptides, such as p47-54 (BMRF1), p138 (BALF2), p55-DNAse (BGLF5), and p65-TK (BXLF1), but without viral capsid (VCA) or nuclear antigen (EBNA) reactivity, gave highest sensitivity (90.4%) and specificity (95.5%) values for NPC diagnosis in both IgG and IgA ELISA. The data support further the notion that EBV-EA reactive IgG and IgA antibodies in NPC patients are directed against distinct conformational and-in part-linear epitopes on EBV-specific proteins, barely recognized in other EBV-related syndromes. The use of a defined native EBV EA-specific antigen opens the way to further improve serological diagnosis of NPC.

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

“…The EA antigen used in this study was produced by 0.3 M salt extractions from purified EA nuclei as described elsewhere (22). Synthetic peptides for the EBNA1 plus VCA p18 ELISA ([EBNA1ϩVCA p18] ELISA) have been described before (7).…”

“…Horseradish peroxidase activity was detected by 3,3Ј,5,5Ј-tetramethylbenzidine (bioMerieux, Boxtel, The Netherlands) and stopped by adding 1 M H 2 SO 4 . The optical density at 450 nm (OD 450 ) was determined and cutoff values (CoVs) were determined by receiver operating characteristic (ROC) analysis (8,22).…”

“…In-house immunoblot assays using recombinant proteins or natural EBV antigen prepared from superinducible HH514 cells were done as described previously (7,19,22). Details and interpretation of immunoblot patterns have been described before (5,7,16,25).…”

“…Details and interpretation of immunoblot patterns have been described before (5,7,16,25). For some experiments we used purified in-house recombinant proteins as described elsewhere (22).…”

“…The combined EBV IgA ELISA and immunoblot assay showed increased sensitivity and specificity and positive predictive value (PPV) and negative predictive value (NPV) of more than 95% (7,8). Because immunoblot studies revealed a diagnostic value of multiple EBV proteins, in particular certain EBV-EA markers, we recently developed a separate IgA EA ELISA using native EA proteins (22). In addition to their role in primary diagnosis, anti-EBV IgA responses, in particular the IgA EA response, also have a distinct role for posttreatment follow-up monitoring, as declining responses correlate with a good prognosis and increasing responses are related to persistence of relapsing tumor (6,16,24).…”