Nonisotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody against DNA-RNA hybrids.
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 degrees C; probe concentration, 0.2 microgram/ml; extent of probe biotinylation, 6.7%; buffer stringency, 2x 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).
Coutlee
F
F
Eudowood Division of Infectious Diseases, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
Yolken
R H
RH
Viscidi
R P
RP
D000818:Animals; D000911:Antibodies, Monoclonal; D001710:Biotin; D015342:DNA Probes; D004926:Escherichia coli / Q000235:genetics; D007124:Immunoenzyme Techniques; D007700:Kinetics; D008832:Microchemistry / Q000379:methods; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012313:RNA / Q000032:analysis*
D000911:Antibodies, Monoclonal; D015342:DNA Probes; D012313:RNA; D001710:Biotin