Vol. 10, No. 5

JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1979, p. 747-751 0095-1137/79/11-0747/05$02.00/0

Enzyme Immunoassay for Detection of Antibody to EpsteinBarr Virus-Specific Early Antigen DAVID J. GRANLUND,lt* PAUL H. LEVINE,2 AND DAVID A. FUCCILLO' Inc., Kensington, Maryland 207951 and National Institutes of Health, Bethesda, Maryland Bionetics, Litton 200142

Received for publication 2 August 1979

Antibody to Epstein-Barr virus-induced early antigen could be measured in using enzyme-linked antibody. The sensitivity of this assay was comparable to that of the indirect immunofluorescent technique. Measurement of early antigen was accomplished on a producer cell line which was specifically treated to block late gene expression. It is now possible to measure Epstein-Barr virusinduced early antigen and viral capsid antigen antibodies by using the same cell line. sera

It has been demonstrated that enzyme-linked antibody can increase the sensitivity of the measurement of Epstein-Barr virus (EBV)-specific viral capsid antigen (VCA) antibody in serum (1; D. J. Granlund, P. H. Levine, and D. A. Fuccillo, submitted for publication). Historically, the measurement of EBV early antigen (EA) has been done with Raji cells. Several observations suggest that Raji cells may not be the best choice. It has been demonstrated that Raji cells have receptors for complement (6) on their surface, and this has led to the development of an assay for the detection of immune complexes in serum. Further, Raji cells have receptors for immunoglobulin G Fc as well as C3b and 03d. Although indirect immunofluorescence has been used successfully for the measurement of EBV-EA in these cells (3), the increased sensitivity of the enzyme-linked antibody method will not allow the use of this system. It has been demonstrated that phosphonoacetic acid (PAA) in the appropriate concentration inhibits EBV-deoxyribonucleic acid synthesis and late gene (VCA) expression (5). It has also been demonstrated that EBV membrane antigen (MA) is inhibited by PAA (2). We confirm that PAA blocks expression of late gene expression in the EBV-productively infected cells B95-8 and P3HR1. Further, we confirn the report of Patel and Menezes (4) that PAA blocks the expression of EA in P3HR1 cells as well as the late antigens. We present data demonstrating that EBV-EA can be measured in B95-8 cells without the interference of nonspecific binding of immunoglobulin G found with Raji cells and that the enzyme-linked antibody system is comparable to indirect irumunofluorescence. t Present address: Biotech Research Laboratories, Rockvile, MD 20852.

MATERIALS AND METHODS Cells used for this study were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (GIBCO Laboratories, Grand Island, N.Y.) and 50 Lg of gentamicin (Microbiological Associates, Bethesda, Md.) per ml. Cells were subcultured when their density reached approximately 1.5 x 106/ml. B95-8 and P3HR1 cells were cultured as usual for control cultures, and parallel cultures received 100 yg of PAA (a gift from Abbott Laboratories, North Chicago, Ill.) per ml. Slides were prepared by washing the cells twice with phosphate-buffered saline before placing 10 ,ll of a cell suspension (107 cells) in each well of multiplewell slides (Bio-Research Glass, Inc., Vineland, N.J.). The slides were forced-air dried at room temperature and fixed in acetone for 10 min. The slides were then stored (-70°C) until used. The technique used for staining the slides was fundamentally as previously reported (1), except that the enzyme-antibody slides were counterstained before reading. Slides were counterstained for 10 s with a solution of (per liter): 5 g of hematoxylin, 50 g of aluminum ammonium sulfate, 0.25 g of sodium periodate, 300 ml of glycerine, and 20 ml of glacial acetic acid, made to 1 liter in distilled water; slides were then rinsed in tap water and mounted with phosphate - buffered saline:glycerol (1:1). PAA-treated cell slides were tested for VCA expression by indirect immunofluorescence and indirect enzyme immunoassay (EIA) by using a VCA-positive, EA-negative serum. In addition, direct enzyme-antibody staining was used to demonstrate EBV-specific antigen expression in producer cells. The conjugate for direct staining was prepared by coupling horseradish peroxidase to the isolated immunoglobulin G from a Burkitt's lymphoma serum. Sera were tested under code. The sera with substantial EBV-EA titers were from patients with infectious mononucleosis, nasopharyngeal carcinoma, or Burkitt's lymphoma. One serial set of samples was analyzed from an individual who developed infectious mononucleosis. 747



RESULTS In B95-8 cells, EA was relatively unaffected by the continuous presence of PAA at 100 ,ug/ ml (Fig. 1). In contrast, VCA and MA were severely depressed by PAA. Less than one cell per thousand was detected as expressing VCA or MA. It was found that 4 to 5 days after the removal of PAA from the medium, both VCA and MA returned to levels indistinguishable from those in the control cultures. P3HR1 cells required longer treatment with PAA to suppress the late gene expression than did B95-8 cells. In three experiments with P3HR1 cells, VCA and MA were effectively suppressed by 7, 9, and 11 100 days of continuous exposure to PAA at lg/ ml. However, EA was suppressed in the same time period. When direct EIA was performed, results were similar to those found by the indirect EIA for EA/VCA. Using PAA-treated B95-8 cells, 40 sera were analyzed for anti-EA antibody (Fig. 2). The two


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Enzyme immunoassay for detection of antibody to Epstein-Barr virus-specific early antigen.

Vol. 10, No. 5 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1979, p. 747-751 0095-1137/79/11-0747/05$02.00/0 Enzyme Immunoassay for Detection of Antibody...
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