Epstein-Barr virus suspension cell assay using in situ hybridization and flow cytometry

✍ Scribed by Jill Crouch; David Leitenberg; Brian R. Smith; John Greg Howe

• Book ID: 101242155
• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 258 KB
• Volume: 29
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(19970901)29:1<50::aid-cyto5>3.0.co;2-h

✦ Synopsis

We have developed a procedure for quantitative assay of Epstein-Barr virus (EBV)-infected cells in suspension in either latent or replicative phase using in situ hybridization and flow cytometry. The cells were hybridized with EBV-specific digoxigenin or biotin-labeled oligonucleotide probes, followed by binding to fluorescein-conjugated anti-digoxigenin or phycoerythrinconjugated streptavidin, respectively. The cells hybridizing to the specific probes were quantitated by flow cytometry. A strong shift in fluorescence intensity (20fold) was observed when the EBV-positive culture cells were hybridized with a specific EBER1 antisense probe. The sensitivity of the assay was at least one positive cell out of 9,000 beyond the normal control mean ؎2 S.D.

We performed two-color in situ hybridization/ flow cytometry using probes to an EBV replication phase-specific mRNA and EBER1 on B95-8 cells in which a small portion (2-4%) of cells induce spontaneously into the replicative phase. In addition, we have developed a method for simultaneous analysis of the cell surface phenotype and EBV nucleic acid content in individual cells. Cytometry 29:50-57,