Poly A-linked colorimetric microtiter plate assay for HIV reverse transcriptase.
An assay for detection of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using poly A linked to microtiter plate with colorimetric detection of incorporated biotin deoxyuridine triphosphate (biotin-dUTP). During the RT reaction, biotin-dUTP was incorporated into oligodeoxythymidylic acid (oligo-dT) which had been hybridized with poly A. At the detection step, horseradish peroxidase conjugated streptavidin was added, followed by the reaction of a colorimetric substrate for this enzyme. This method was contrasted with the two standard isotopic RT assays. There was excellent correlation between the colorimetric RT assay and each of two isotopic RT assays for both detection and quantification of avian myoblastosis virus reverse transcriptase (AMV-RT) and of HIV RT in human lymphocytes infected in vitro with HIV-1. The total assay required for performing the colorimetric assay, including the RT reaction, was 40 min.
Suzuki
K
K
Biomedical Research Center, Olympus Corporation, East Setauket, NY.
Craddock
B P
BP
Okamoto
N
N
Kano
T
T
Steigbigel
R T
RT
D001710:Biotin; D003124:Colorimetry; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives; D004797:Enzyme-Linked Immunosorbent Assay; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000302:isolation & purification / Q000502:physiology; D006801:Humans; D007202:Indicators and Reagents; D007963:Leukocytes, Mononuclear / Q000382:microbiology*; D011061:Poly A; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012190:Retroviridae / Q000201:enzymology; D012680:Sensitivity and Specificity
D003856:Deoxyuracil Nucleotides; D007202:Indicators and Reagents; C070202:digoxigenin-11-deoxyuridine triphosphate; D011061:Poly A; D001710:Biotin; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004076:Digoxigenin