Evaluation of Antibodies to the Epstein-Barr Virus Immediate Early Gene Product ZEBRA by a New Enzyme-Linked Immunosorbent Assay

Abstract

For the serodiagnosis of Epstein-Barr virus (EBV) infections, we have developed a new enzyme-linked immunosorbent assay (ELISA) for antibodies to the ZEBRA product of EBV immediate early gene BZLF1. ZEBRA protein fused with glutathione-S-transferase (GST) was expressed in Escherichia coli and purified by affinity chromatography with glutathione-Sepharose 4B. An ELISA sandwich capture system was constructed with the GST-ZEBRA immobilized on plastic microtiter plates which had been coated with a mouse monoclonal antibody to GST. ZEBRA-IgG antibodies in patients’ sera with chronic active EBV infection (CAEBV) and infectious mononucleosis (IM) had, respectively, very high and high titers. Anti-ZEBRA antibodies were also detected at low titers in sera of some healthy controls. ZEBRA-IgM antibodies were detected in sera of patients with IM and CAEBV but not in sera of healthy controls. In sera of patients with CAEBV, the titers of IgG antibodies to ZEBRA correlated with the antibody titers to early antigens obtained with an immunofluorescence assay, but not to EBV nuclear antigens. This ELISA is a useful diagnostic and prognostic test for EBV infection.

Evaluation of an Immunofiltration Assay That Detects Immunoglobulin M Antibodies against the ZEBRA Protein for the Diagnosis of Epstein-Barr Virus Infectious Mononucleosis in Immunocompetent Patients

“…In effect, 42 of 47 sera (89.3%) displaying a antibody profile typical of an acute primary EBV infection (VCA IgG ϩ or VCA IgG Ϫ , VCA IgM ϩ , and EBNA-1 IgG Ϫ ) gave a positive result in the IMFA. This figure is in contrast with that (14%) previously reported by another group (18). The most likely explanation for this discrepancy is the different natures of both the antigens and the immunoassay formats employed (a prokaryotic recombinant ZEBRA protein and a sandwich capture enzyme-linked immunosorbent assay were used in the abovementioned study, whereas a combination of immunogenic ZEBRA-derived synthetic peptides were employed in the evaluated IMFA in this study).…”

“…To our knowledge, there are no published data in relation to the clinical value of assessing the presence of IgMs to ZEBRA for the diagnosis of EBV IM except for a study evaluating the performance of a capture enzyme-linked immunosorbent assay that detects antibodies to a recombinant ZEBRA protein (18). The ZEBRA immunofiltration test was found to be highly sensitive (92.5%) and specific (97.3%) for the diagnosis of EBV IM.…”

“…ZEBRA is expressed during the lytic cycle in EBV-permissive cells and plays a critical role in transactivating several immediate early, early, and late EBV genes (5). Antibodies against ZEBRA are produced during primary EBV infection (11,15,18), and thus, the detection of ZEBRA-specific IgMs may allow an early diagnosis of EBV IM. In the present study, we evaluated a rapid and easy-to-perform immunofiltration assay (IMFA) detecting IgMs to the EBV ZEBRA protein for the biological diagnosis of IM in immunocompetent patients.…”

Development of a Time-Resolved Fluorescence Immunoassay for Epstein–Barr Virus Zta IgA Antibodies in Human Serum

“…As described above, the ZEBRA antibody is a sensitive and specific serological marker for detecting EBV-associated disease because it is rare among normal samples (6,27). High levels of anti-ZEBRA antibody could be used not only as a biological marker in the follow-up of NPC and non-Hodgkin's lymphoma patients but also for the prognosis of EBV infectious diseases (22)(23)(24). It has been reported that IgA antibodies could more effectively indicate the risk of NPC (2).…”

Chicken Antibodies: A Useful Tool for Antigen Capture ELISA to Detect Bovine Leukaemia Virus without Cross-reaction with Other Mammalian Antibodies

“…In this way, epitopes on the protein are presented to antibodies in the least obstructed and most desirable way. Several attempts have been made to immobilize pure GST to the bottom of the microwell (Cartwright et al, 1995), covalently link GST to another protein such as haemoglobin (Murray et al, 1998) or casein (Sehr et al, 2001) or to directly bind mammalian antibodies against GST (monoclonal or polyclonal) to the bottom of the plate (Kilty et al, 1998;Yamauchi et al, 1998).…”