Comparable sensitivities for detection of HIV-1 reverse transcriptase (RT) and other polymerases by RT assays requiring no radioisotopic materials.
An improved non-radioisotopic (Non-RI) reverse transcriptase (RT) assay with a template-primer-immobilized microtiter plate is described, which has greater sensitivity than the former Non-RI RT assay previously described. Non-RI and commercially available non-radioactive (Non-RA) RT assays were compared for their ability to detect various polymerases. Two RTs from Rous-associated virus 2 (RAV-2) and avian myeloblastosis virus (AMV), one polymerase from Escherichia coli (Pol-I) and one recombinant RT of human immunodeficiency virus type 1 (HIV-1) were assessed. Two HIV-1 samples in a culture supernatant and pelleted virion suspended in Triton X-100 solution were measured. The Non-RI RT assay was one hundred times more sensitive by RAV-2 and Pol-I polymerases, and one thousand times more sensitive by the Non-RA assay than by the AMV RT. The Non-RI RT assay was 10, 16 and 64 times more sensitive than the Non-RA assay for measuring recombinant HIV-1 RT, pelleted virus and virus suspended in culture medium, respectively. To explain the discrepancy, it is shown that free biotin, such as in culture medium, disturbs the assay system of the Non-RA RT assay, but not the Non-RI assay. The present assay can be used to clarify the inhibitory mechanism of an anti-HIV-1 substance.
Sano
K
K
Department of Microbiology, Osaka Medical College, Japan.
Odawara
F
F
Nakano
T
T
Morimatsu
S
S
Nakamura
T
T
Saitoh
Y
Y
Jiang
Y
Y
Misaki
H
H
Sakai
Y
Y
Nakai
M
M
D000818:Animals; D001354:Avian Leukosis Virus / Q000201:enzymology; D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D001681:Biological Assay / Q000379:methods*; D001710:Biotin; D002460:Cell Line; D004259:DNA-Directed DNA Polymerase / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology; D054303:HIV Reverse Transcriptase; D006801:Humans; D012194:RNA-Directed DNA Polymerase / Q000032:analysis*; D011868:Radioisotopes; D012680:Sensitivity and Specificity
D011868:Radioisotopes; D001710:Biotin; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase