Chemiluminescent enzyme-linked immunoassay for reverse transcriptase, illustrated by detection of HIV reverse transcriptase.
A chemiluminescent assay for reverse transcriptase (RT) of the human immunodeficiency virus 1 was developed using biotin-labeled oligodeoxythymidylic acid (biotin oligo-dT) and digoxigenin-deoxyuridine triphosphate instead of tritiated thymidine triphosphate. After the RT reaction, the newly polymerized strand from biotin oligo-dT contained digoxigenin labels. This nucleotide was bound to a streptavidin-coated microtiter plate by the reaction to biotin. At the detection step, an alkaline phosphatase-conjugated antibody to digoxigenin was added, followed by the reaction of a chemiluminescent substrate for this enzyme. This method shows very close correlation with the isotopic assay using purified avian myeloblastosis virus reverse transcriptase (RT). This assay was also compared with the isotopic RT assay using lymphocytes infected in vitro with HTLV-IIIB and again demonstrated a close correlation. The total assay time after the RT reaction step was less than 100 min.
Suzuki
K
K
Biomedical Research Center, Olympus Corporation, East Setauket, New York 11733.
Craddock
B P
BP
Kano
T
T
Steigbigel
R T
RT
D000469:Alkaline Phosphatase; D000906:Antibodies; D001426:Bacterial Proteins; D001710:Biotin; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015658:HIV Infections / Q000097:blood / Q000201:enzymology; D054303:HIV Reverse Transcriptase; D006801:Humans; D008163:Luminescent Measurements; D008214:Lymphocytes / Q000201:enzymology / Q000382:microbiology; D009838:Oligodeoxyribonucleotides; D011061:Poly A; D012194:RNA-Directed DNA Polymerase / Q000032:analysis* / Q000097:blood; D012015:Reference Standards; D012680:Sensitivity and Specificity; D019809:Streptavidin; D013997:Time Factors
D000906:Antibodies; D001426:Bacterial Proteins; D003856:Deoxyuracil Nucleotides; D009838:Oligodeoxyribonucleotides; C070202:digoxigenin-11-deoxyuridine triphosphate; C027903:oligo (dT); D011061:Poly A; D001710:Biotin; D019809:Streptavidin; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D000469:Alkaline Phosphatase; D004076:Digoxigenin