[Non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridine-triphosphate and a primer-immobilized microtiter plate: application for detection and identification of isolated retroviruses from HIV-1-seropositive hemophiliac patients].
Non-radioisotopic reverse transcriptase assay (Non-RTA) was successfully applied for detection and identification of the retroviruses isolated from peripheral mononuclear cells from eight HIV-1-seropositive hemophiliac patients. Of 40 samples, 36 (90%) were consistent in detection between Non-RI and RI RTA. Four samples which showed RT activities slightly above the cutoff level of RI RTA were not detected by Non-RI RTA. Non-specific RT-inhibitors in the culture supernatant decreased the sensitivity of Non-RI RTA more significantly than that of RI RTA. It was demonstrated that higher concentrations (> 20%) of fetal calf serum in RPMI-1640 culture medium inhibited the hybridization of poly rA template with immobilized primer, resulting in reducing the sensitivity of Non-RI RTA. Then we identified the isolated retroviruses using specific RT-inhibiting (RTI) antibodies against HIV-1 and HIV-2. HIV-1 RTI antibody specifically inhibited RTs of isolated retroviruses and HIV-1 strain, LAV-1, but not HIV-2 and Rous-associated virus 2 (RVA-2) RT. Conversely, HIV-2 RTI antibody specifically inhibited HIV-2 RT, but not HIV-1 and RAV-2 RT. These findings agreed with previously reported results showing the type-specificity of HIV-1 and -2 RTI antibodies, and suggest the possibility that isolated retroviruses could be identified by these antibodies.
Urabe
T
T
Department of Microbiology, Osaka Medical College.
Sano
K
K
Odawara
F
F
Otake
T
T
Mori
H
H
Morimoto
M
M
Okubo
S
S
Ishikawa
K
K
Nakai
M
M
D001710:Biotin; D003856:Deoxyuracil Nucleotides; D006679:HIV Seropositivity / Q000382:microbiology*; D015497:HIV-1 / Q000302:isolation & purification; D006467:Hemophilia A / Q000382:microbiology*; D006801:Humans; D008828:Microbiological Techniques; D009000:Monocytes / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase / Q000032:analysis*; D012190:Retroviridae / Q000302:isolation & purification*
D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate; D001710:Biotin; D012194:RNA-Directed DNA Polymerase