EPSTEIN-BARR-VIRUS SUSPENSION CELL ASSAY USING IN-SITU HYBRIDIZATION AND FLOW-CYTOMETRY

We have developed a procedure for quantitative assay of Epstein-Barr v irus (EBV)-infected cells in suspension in either latent or replicativ e phase using in situ hybridization and flow cytometry, The cells were hybridized With EBV-specific digoxigenin or biotin-labeled oligonucle otide probes, followed by binding to fluorescein-conjugated anti-digox igenin or phycoerythrin-conjugated streptavidin, respectively, The cel ls hybridizing to the specific probes were quantitated bq flow cytomet ry, A strong shift in fluorescence intensity (20-fold) mas observed wh en the EBV-positive culture cells were hybridized with a specific EBER 1 antisense probe. The sensitivity of the assay was at least one posit ive cell out of 9,000 beyond the normal control mean +/-2 S.D. We perf ormed two-color in situ hybridization/flow cytometry using probes to a n EBV replication phase-specific mRNA and EBER1 on B95-8 cells in whic h a small portion (2-4%) of cells induce spontaneously into the replic ative phase, In addition, we have developed a method for simultaneous analysis of the cell surface phenotype and EBV nucleic acid content ir e individual cells. (C) 1997 Wiley-Liss, Inc.