Epstein-Barr virus suspension cell assay using in situ hybridization and flow cytometry.
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 phycoerythrin-conjugated streptavidin, respectively. The cells hybridizing to the specific probes were quantitated by flow cytometry. A strong shift in fluorescence intensity (20-fold) 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.
Crouch
J
J
Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8035, USA.
Leitenberg
D
D
Smith
B R
BR
Howe
J G
JG
D002469:Cell Separation / Q000379:methods; D002472:Cell Transformation, Viral; D005434:Flow Cytometry; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D017403:In Situ Hybridization; D019169:Jurkat Cells; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014776:Virus Cultivation
C099537:Epstein-Barr virus encoded RNA 1; C106732:Epstein-Barr virus encoded RNA 2; D012333:RNA, Messenger; D012367:RNA, Viral