Diffuse Early Antigen Research Articles

Serodiagnosis of infectious mononucleosis by using recombinant Epstein-Barr virus antigens and enzyme-linked immunosorbent assay technology

Four recombinant, diagnostically useful Epstein-Barr virus (EBV) proteins representative of the viral capsid antigen (p150), diffuse early antigen (p54), the major DNA-binding protein (p138), and the EBV nuclear antigen (p72) (W. Hinderer, H. Nebel-Schickel, H.H. Sonneborn, M. Motz, R. Kühbeck, and H. Wolf, J. Exp. Clin. Cancer Res. 7[Suppl.]:132, 1988) were used to set up individual enzyme-linked immunosorbent assays (ELISAs) for the qualitative and quantitative detection of immunoglobulin M (IgM) and IgG antibodies. In direct comparison with results obtained by standard immunofluorescence or immunoperoxidase assays, it was then shown that the recombinant EBV ELISAs provide the means for specific and sensitive serodiagnosis of infectious mononucleosis (IM) caused by EBV. The most useful markers in sera from such patients proved to be IgM antibodies against p54, p138, and p150. Additional positive markers for recent or ongoing IM apparently were IgG antibodies against p54 and p138. In contrast, anti-p72 IgG had a high preference for sera from healthy blood donors and, therefore, can be considered indicative of past exposure to the virus. Altogether, the individual ELISAs proved to be as specific and at least as sensitive for the diagnosis of IM as the currently available standard techniques are. Moreover, our findings suggest that, by combining individual test antigens, a workable ELISA system consisting of three assays (IgM against p54, p138, and p150; IgG against p54 and p138; and IgG against p72) can be established for the standardized rapid diagnosis of acute EBV infections.

Comparison of the classical immunofluorescent antibody tests for epstein‐barr virus with commercially available test kits

Twenty reference sera with previously reported titers plus 10 normal seropositive and 10 seronegative sera were used to determine which assay for the detection of antibodies to Epstein-Barr virus should be utilized in our laboratory. By three commercially available test systems and by slides prepared in our laboratory, all techniques detected the same number of samples positive for IgG antibodies to viral capsid antigen (VCA) and Epstein-Barr virus-determined nuclear antigen (EBNA), but the geometric mean titers (GMTs) varied. Major discrepancies arose in the sensitivity of detection of early antigen-diffuse (EA-D) and -diffuse and restricted (EA-DR). Slides prepared in our laboratory gave the best results, detecting 12 of 12 (100%). Slides prepared according to Gull and Hillcrest detected 11 of 12 (92%), and those prepared according to Zeus detected 9 of 12 (75%). From these data, we conclude that all of these methods for detection of VCA and EBNA are adequate. However, in determining antibody levels to EA-D and EA-DR, great care should be taken as to the source of the slide and specificity and sensitivity.

Synthetic peptide derived from the epstein‐barr virus encoded early diffuse antigen (ea‐d) reactive with human antibodies

AbstractPrimary infection with Epstein‐Barr virus (EBV) and reactivation of latent virus are associated with increased antibody titers against the diffuse early antigen (EA‐D). In order to better define the antigenic epitopes recognized by antibodies from patients with infectious mononucleosis (IM) and with other disease states, a series of synthetic peptides were prepared based on the DNA sequence encoding the EA‐D molecule. One synthetic peptide (K7b) was reactive with the majority of sera from patients with acute IM. Anti‐K7b activity was most readily detected among IgM and IgA antibodies and to a lesser extent among IgG antibodies. In contrast, significant elevations of anti‐K7b activity were observed in less than 5% of healthy adults. Serial analysis of samples from individuals prior to and after exposure to EBV demonstrated increased anti‐K7b reactivity associated with the symptoms of acute IM. Elevated anti‐peptide K7b titers also were found in sera of patients with nasopharyngeal carcinoma and with Sjogren's syndrome (an autoimmune disease involving the salivary glands). Four different synthetic peptides from other regions of the EA‐D molecule were not reactive with antibodies from these patients nor from IM patients. These results suggest that peptide K7b defines an antigenic epitope recognized during primary EBV infection and during viral reactivation occurring in patients with autoimmune and neoplastic disease.

A second Epstein-Barr virus early antigen gene in BamHI fragment M encodes a 48- to 50-kilodalton nuclear protein.

We used antiserum raised against the bacterially synthesized product of one of the open reading frames in Epstein-Barr virus (EBV) BamHI fragment M to demonstrate that this reading frame (BMRF1) codes for a nuclear protein of the diffuse early antigen (EA) class. In indirect immunofluorescence assays, the rabbit anti-BMRF1 antiserum gave nuclear staining in approximately 5% of Raji cells which had been treated with sodium butyrate, and positive fluorescence was observed in both acetone- and methanol-fixed cells. Uninduced Raji cultures contained less than 0.1% positive cells regardless of whether indirect immunofluorescence or anti-complement immunofluorescence was used. In immunoblot analyses, the rabbit serum identified a family of polypeptides of 46 to 55 kilodaltons (kDa) in total protein extracts from B95-8 cells or from butyrate-induced Raji cells. In both cell types, the dominant polypeptides were the 48- and 50-kDa species. This same family of polypeptides was identified when the immunoblots were reacted with the R3 monoclonal antibody, and we concluded that this antibody also recognized the product of the BMRF1 open reading frame. Fibroblast cell lines containing EBV BamHI fragment M were established by cotransfection of baby hamster kidney cells with BamHI-M and the gene for neomycin resistance. Aminoglycoside G418-resistant colonies which showed evidence for EBV antigen expression in immunofluorescence assays were selected, and clonal cell lines were established. After 3 to 4 months of passaging, constitutive synthesis of EA was no longer detectable in these cell lines either by immunofluorescence or by immunoblot analysis. However, in the one cell line examined, synthesis of the 48- to 50-kDa EA was induced by treatment of the culture with sodium butyrate. Thus, the regulation of expression of this EA in transfected fibroblasts is analogous to that seen in Raji lymphoblasts. We showed previously that BamHI fragment M also contains the coding sequences for a 60-kDa nuclear EA, and hence BamHI-M encodes two separate components of the diffuse EA complex.

Functional mapping of the Epstein-Barr virus genome: Identification of sites coding for the restricted early antigen, the diffuse early antigen, and the nuclear antigen

Attempts were made to functionally map antigenic expression of the Epstein-Barr virus (EBV) to specific regions on the EBV genome, using the B95-8 strain. Experiments were performed to map the expression of early antigen (EA), both restricted and diffuse (R and D, respectively), and the EBV nuclear antigen (EBNA), using intact B95-8 DNA, cloned BamHI fragments or Charon 4A fragments. DNA preparations were microinjected into two EBV genome-negative epithelial tumor cell lines. Expression of EBV antigens was monitored using precharacterized human sera, as well as monoclonal antibodies to EA-R and EA-D. The data suggest that EA-R maps to the BamHI H fragment, and EA-D maps to the Charon 4A fragment 7. A previous report that BamHI K is associated with the expression of a nuclear neoantigen tentatively identified as EBNA ( W. P. Summers, E. A. Grogan, D. Shedd, M. Robert, C. R. Liu, and G. Miller, Proc. Nat. Acad. Sci. USA 79, 5688–5692, 1982 ) was also confirmed.

Antibody response in cattle and rabbits to early antigens of malignant catarrhal fever virus in cultured cells

Cytosine arabinoside (Ara-C) inhibited the production of late antigens and of infectious virus in monolayers of bovine kidney cells infected with the high-passage, WC-11 strain of malignant catarrhal fever virus. Early antigens were not affected. Using hyperimmune and acute-phase sera from cattle and rabbits in indirect immunofluorescence tests, it was shown that Ara-C treated cultures contained two early antigens; one was diffuse and distributed throughout the cells, the other was particulate and intranuclear. Antibody to early antigens developed later and attained lower titres in infected animals, especially rabbits; only hyperimmune sera reacted with the diffuse early antigen.