Colorimetric reverse transcriptase assay for HIV-1.
A colorimetric assay for reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) was developed using a double labelled (biotin and digoxigenin) deoxyuridine triphosphate mixture instead of tritiated thymidine triphosphate. After the RT reaction, the newly polymerized strand from oligodeoxythymidylic acid (oligo-dT) contained both biotin and digoxigenin labels. This nucleotide was bound to streptavidin-magnetic beads by the reaction to biotin. At the detection step, an alkaline phosphatase conjugated antibody to digoxigenin was added, followed by the reaction of a colorimetric substrate for this enzyme. This RT assay was comparable to the isotopic RT assay using purified AMV-RT and two strains of HIV grown in cell lines. In addition it was equivalent to the isotopic RT assay for analysis of the time course of in vitro infection of human peripheral blood lymphocytes by HIV-1. The total assay time after the RT reaction step was less than one hour.
Suzuki
K
K
Biomedical Research Center, Olympus Corporation, East Setauket, NY 11733.
Craddock
B P
BP
Kano
T
T
Steigbigel
R T
RT
D001710:Biotin; D002460:Cell Line; D003124:Colorimetry / Q000379:methods* / Q000592:standards; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin; D005069:Evaluation Studies as Topic; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D012194:RNA-Directed DNA Polymerase / Q000032:analysis* / Q000592:standards; D012015:Reference Standards; D013942:Thymine Nucleotides; D014316:Tritium; D014773:Virology / Q000379:methods* / Q000592:standards
D003856:Deoxyuracil Nucleotides; D013942:Thymine Nucleotides; D014316:Tritium; C027078:deoxyuridine triphosphate; D001710:Biotin; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004076:Digoxigenin; C024157:thymidine 5'-triphosphate