Detection of transcripts of human papillomaviruses 16 and 18 in cancer-derived cell lines and cervical biopsies by enzyme immunoassay for DNA-RNA hybrids following solution hybridization

F. Coutlee, K. V. Shah, J. S. Rader, J. L. Currie, Raphael P Viscidi

Research output: Contribution to journalArticle

Abstract

mRNAs of human papillomaviruses (HPV) 16 and 18 were detected in cancer-derived cell lines and genital tract biopsy specimens by a novel hybridization assay. Biotinylated whole genomic HPV DNA probes were hybridized in solution to extracted total nucleic acids. Hybrids between the labeled probes and RNA transcripts were captured on a microplate coated with an antibiotin antibody. Bound hybrids were incubated with a β-galactosidase-labeled monoclonal antibody to DNA-RNA hybrids and measured by the addition of a fluorogenic substrate. HPV 18 and HPV 16 mRNAs were detected in nucleic acids from 2.3 x 103 HeLa cells and 104 SiHa cells, respectively. The specificity of the assay for mRNA was demonstrated by the low reactivity of nucleic acids from SiHa cells after treatment with T1 RNase and by the selective reactivity of cellular nucleic acids which bound to an oligo(dT) column. With HPV 16 subgenomic probes, E6-E7 transcripts but not L1-L2 transcripts were detected in SiHa cells. Tests of 58 biopsy specimens from 31 patients showed that the detection of HPV 16 and HPV 18 transcripts in tissue specimens was feasible. Analysis of biopsy specimens with subgenomic probes revealed HPV 16 E6-E7 transcripts in all specimens that reacted with the whole genomic probe, while L1-L2 transcripts were found infrequently.

Original languageEnglish (US)
Pages (from-to)968-974
Number of pages7
JournalJournal of Clinical Microbiology
Volume29
Issue number5
StatePublished - 1991
Externally publishedYes

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Human papillomavirus 18
Human papillomavirus 16
Immunoenzyme Techniques
Nucleic Acids
RNA
Biopsy
Cell Line
DNA
Neoplasms
Messenger RNA
Galactosidases
Ribonuclease T1
RNA Probes
DNA Probes
Fluorescent Dyes
HeLa Cells
Monoclonal Antibodies
Antibodies

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology

Cite this

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title = "Detection of transcripts of human papillomaviruses 16 and 18 in cancer-derived cell lines and cervical biopsies by enzyme immunoassay for DNA-RNA hybrids following solution hybridization",
abstract = "mRNAs of human papillomaviruses (HPV) 16 and 18 were detected in cancer-derived cell lines and genital tract biopsy specimens by a novel hybridization assay. Biotinylated whole genomic HPV DNA probes were hybridized in solution to extracted total nucleic acids. Hybrids between the labeled probes and RNA transcripts were captured on a microplate coated with an antibiotin antibody. Bound hybrids were incubated with a β-galactosidase-labeled monoclonal antibody to DNA-RNA hybrids and measured by the addition of a fluorogenic substrate. HPV 18 and HPV 16 mRNAs were detected in nucleic acids from 2.3 x 103 HeLa cells and 104 SiHa cells, respectively. The specificity of the assay for mRNA was demonstrated by the low reactivity of nucleic acids from SiHa cells after treatment with T1 RNase and by the selective reactivity of cellular nucleic acids which bound to an oligo(dT) column. With HPV 16 subgenomic probes, E6-E7 transcripts but not L1-L2 transcripts were detected in SiHa cells. Tests of 58 biopsy specimens from 31 patients showed that the detection of HPV 16 and HPV 18 transcripts in tissue specimens was feasible. Analysis of biopsy specimens with subgenomic probes revealed HPV 16 E6-E7 transcripts in all specimens that reacted with the whole genomic probe, while L1-L2 transcripts were found infrequently.",
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T1 - Detection of transcripts of human papillomaviruses 16 and 18 in cancer-derived cell lines and cervical biopsies by enzyme immunoassay for DNA-RNA hybrids following solution hybridization

AU - Coutlee, F.

AU - Shah, K. V.

AU - Rader, J. S.

AU - Currie, J. L.

AU - Viscidi, Raphael P

PY - 1991

Y1 - 1991

N2 - mRNAs of human papillomaviruses (HPV) 16 and 18 were detected in cancer-derived cell lines and genital tract biopsy specimens by a novel hybridization assay. Biotinylated whole genomic HPV DNA probes were hybridized in solution to extracted total nucleic acids. Hybrids between the labeled probes and RNA transcripts were captured on a microplate coated with an antibiotin antibody. Bound hybrids were incubated with a β-galactosidase-labeled monoclonal antibody to DNA-RNA hybrids and measured by the addition of a fluorogenic substrate. HPV 18 and HPV 16 mRNAs were detected in nucleic acids from 2.3 x 103 HeLa cells and 104 SiHa cells, respectively. The specificity of the assay for mRNA was demonstrated by the low reactivity of nucleic acids from SiHa cells after treatment with T1 RNase and by the selective reactivity of cellular nucleic acids which bound to an oligo(dT) column. With HPV 16 subgenomic probes, E6-E7 transcripts but not L1-L2 transcripts were detected in SiHa cells. Tests of 58 biopsy specimens from 31 patients showed that the detection of HPV 16 and HPV 18 transcripts in tissue specimens was feasible. Analysis of biopsy specimens with subgenomic probes revealed HPV 16 E6-E7 transcripts in all specimens that reacted with the whole genomic probe, while L1-L2 transcripts were found infrequently.

AB - mRNAs of human papillomaviruses (HPV) 16 and 18 were detected in cancer-derived cell lines and genital tract biopsy specimens by a novel hybridization assay. Biotinylated whole genomic HPV DNA probes were hybridized in solution to extracted total nucleic acids. Hybrids between the labeled probes and RNA transcripts were captured on a microplate coated with an antibiotin antibody. Bound hybrids were incubated with a β-galactosidase-labeled monoclonal antibody to DNA-RNA hybrids and measured by the addition of a fluorogenic substrate. HPV 18 and HPV 16 mRNAs were detected in nucleic acids from 2.3 x 103 HeLa cells and 104 SiHa cells, respectively. The specificity of the assay for mRNA was demonstrated by the low reactivity of nucleic acids from SiHa cells after treatment with T1 RNase and by the selective reactivity of cellular nucleic acids which bound to an oligo(dT) column. With HPV 16 subgenomic probes, E6-E7 transcripts but not L1-L2 transcripts were detected in SiHa cells. Tests of 58 biopsy specimens from 31 patients showed that the detection of HPV 16 and HPV 18 transcripts in tissue specimens was feasible. Analysis of biopsy specimens with subgenomic probes revealed HPV 16 E6-E7 transcripts in all specimens that reacted with the whole genomic probe, while L1-L2 transcripts were found infrequently.

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