Nonisotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody against DNA-RNA hybrids

Research output: Contribution to journalArticle

Abstract

A sensitive nonisotopic solution hybridization assay for detection of RNA is described and characterized using a pSP65 plasmid model system. The assay procedure is based on a hybridization reaction in solution between a biotinylated DNA probe and a target RNA. The biotin-labeled hybrids are captured on a microtiter plate coated with an antibody to biotin. Bound DNA-RNA hybrids are detected by an immunoreaction with an enzyme-labeled monoclonal antibody specifically directed against DNA-RNA heteropolymers and the hybrids are quantitatively measured with the addition of a fluorogenic substrate. Optimal conditions under which to perform the assay were hybridization time, 1000 min; temperature, 75°C; probe concentration, 0.2 μg/ml; extent of probe biotinylation, 6.7%; buffer stringency, 2× SSC. A bisulfite-modified DNA probe was compared to nick-translated probes synthesized with reporter groups of different lengths (bio-11-dUTP or bio-19-dUTP). All probes could detect 10 pg/ml of target RNA. The presence of nonhomologous DNA or RNA sequences reduced the sensitivity of RNA detection by one half-log to 32 pg/ml (1.6 pg/assay).

Original languageEnglish (US)
Pages (from-to)153-162
Number of pages10
JournalAnalytical biochemistry
Volume181
Issue number1
DOIs
StatePublished - Aug 15 1989

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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