Poly A-linked colorimetric microtiter plate assay for HIV reverse transcriptase

Suzuki, Kazuo; Craddock, Barbara P.; Okamoto, Naoaki; Kano, Tokio; Steigbigel, Roy T.

doi:10.1016/0166-0934(93)90054-u

Cited by 85 publications

(46 citation statements)

References 11 publications

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“…However, an isotopic assay is time-consuming and restricted to the use and disposal of radioactive material. Recently, alternative methods for measuring RT activity of HIV were developed using non-isotopically labeled nucleoside triphosphates such as BrdUTP or biotin-dUTP [8,9,11]. In Table 2 comparing PAC-RTA and the conventional non-RI RT assay kit with the same RT reaction time, PAC-RTA measured the detection limit of FIV Petaluma strain RT activity about 10 times better than the conventional kit.…”

Section: Discussionmentioning

confidence: 99%

“…Poly A-linked colorimetric RT assay (PAC-RTA): PAC-RTA was performed using the method described by Suzuki et al [9] with slight modifications. Briefly, wells were prepared prior to the assay by adding 50 µl of 80 µg/ml in Eagle's minimum essential medium supplemented with 10% inactivated fetal calf serum (FCS) and antibiotics.…”

Section: Methodsmentioning

confidence: 99%

“…However, the conventional isotopic RT assay requires laborintensive procedures and is becoming increasingly restrictive due to the use of radioactive materials. Recently, alternative methods for the RT assay using non-isotopic agents were developed [8,9,11]. We used the poly A-linked colorimetric reverse transcriptase assay (PAC-RTA) described by Suzuki et al [9] for measurement of HIV RT activity.…”

mentioning

confidence: 99%

“…Recently, alternative methods for the RT assay using non-isotopic agents were developed [8,9,11]. We used the poly A-linked colorimetric reverse transcriptase assay (PAC-RTA) described by Suzuki et al [9] for measurement of HIV RT activity. Since the reverse transcriptase of FIV is very similar to the HIV RT in template specificity and requirements for Mg 2+ [6], we applied the method of PAC-RTA to FIV studies.…”

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confidence: 99%

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Measurement of Reverse Transcriptase of Feline Immunodeficiency Virus by Poly A-Linked Colorimetric Assay.

Saito

Suzuki

Imai

et al. 1997

The Journal of Veterinary Medical Science

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ABSTRACT. The method of the poly A-linked colorimetric reverse transcriptase assay (PAC-RTA) was developed and evaluated for the measurement of Mg 2+ -dependent reverse transcriptase (RT) activity of feline immunodeficiency virus (FIV). PAC-RTA was first evaluated for the detection of RT activity in the culture supernatant of FIV Petaluma strain. The detection limit of RT activity by PAC-RTA was about 10-fold better than that by the conventional non-radioisotopic RT assay kit. Then, PAC-RTA was evaluated for the indication of FIV isolation from cats naturally infected with FIV. FIV was isolated from peripheral blood mononuclear cells of 9 FIV-seropositive cats. The time course appearance of RT activity measured by PAC-RTA corresponded with the analysis of FIV antigen expression by indirect immunofluorescence. Finally, PAC-RTA evaluated the drug susceptibility of FIV. MYA-1 cells (feline T-lymphoblastoid cells) were infected with FIV and were cultured in the presence of various concentrations of anti-human immunodeficiency virus agents such as azidothymidine (AZT) or dextran sulfate. An inverse relationship between the RT activities and the concentrations of these agents in the culture supernatant was confirmed by PAC-RTA. PAC-RTA is easy to perform without using radioactive materials, and one plate can handle 96 samples at one time. By monitoring the RT activity, this assay is a useful method for FIV studies such as viral replication and drug susceptibility. .25 mM MgCl 2 , 62.5 mM Tris pH7.8, 1.25 mM dithiothreitol, and 2.5 µg/ml oligo dT 12-18 (Pharmacia LKB, Uppsala, Sweden)] in microtiter wells. The reaction was carried out at 37°C for 1-15 hr and then washed 3 times with the wash buffer. After the washings, 100 µl of horseradish peroxidase conjugated streptavidin (GIBCO BRL, Gaithersburg, Maryland, U.S.A.) diluted 1:5,000 in TBSE buffer (10 mM Tris pH 7.5, 0.15 M NaCl, 1 mM EDTA and 1% BSA) was added to each well. After the plate was incubated for 20 min at 37°C, the free conjugate was removed by washing 4 times with the wash buffer. As a final step, 50 µl of 3,3',5,5'-tetramethylbenzidine (TMB; Kirkegaard and Perry Laboratories, Inc., U.S.A.) was added to each well. The plate was incubated at room temperature for 15 min. The reactions were stopped by the addition of 50 µl of 1N H 2 SO 4 . The plate was read at 450 nm with a plate reader. All assays were performed in duplicate. Virus: FIV Petaluma strain was used in this study. Crandell feline kidney (CrFK) cells persistently infected with the Petaluma strain (CrFK/Petaluma) [12] were grown

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Measurement of Reverse Transcriptase of Feline Immunodeficiency Virus by Poly A-Linked Colorimetric Assay.

Saito

Suzuki

Imai

et al. 1997

The Journal of Veterinary Medical Science

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“…After the wells were washed three times with wash buffer (10 mM Tris-HCl pH 7.5, 0.15 M NaCl, 1mM EDTA and 0.01% Tween 20), 10 µl of the supernatant from the FIV culture was added to each well with 50 µl of reaction buffer (4.2 µM biotin-dUTP, 8.4 µM TTP, 25 mM KCl, 6.25 mM MgCl 2 62.5 mM Tris pH 7.8, 1.25 mM dithiothreitol and 2.5 µg/ml oligo dT [12][13][14][15][16][17][18] ). The plate was incubated at 37°C for 15 hr and washed with the wash buffer.…”

mentioning

confidence: 99%

Quantitative Measurement of Antibody Inhibiting Reverse Transcriptase Activity in Cats Naturally Infected with Feline Immunodeficiency Virus.

Saito

Suzuki

Imai

et al. 1997

The Journal of Veterinary Medical Science

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ABSTRACT. Quantitative measurement of reverse transcriptase-inhibiting (RTI) antibodies in Japanese household cats naturally infected with feline immunodeficiency virus (FIV) was performed by poly A-linked colorimetric reverse transcriptase assay (PAC-RTA). Eight FIV-seropositive plasma samples were diluted twofold from 1:10 to 1:160 and incubated with FIV RT. Fifty percent RTI activity (RTI 50 ) was calculated from a dose response PAC-RTA curve. The plasma of FIV-seropositive cats showed different RTI activities against two Japanese isolates and Petaluma strain. Six of eight plasma samples showed RTI activities against the Japanese isolates (subtype B), but only one showed RTI activity against Petaluma strain (subtype A). It is important to use the appropriate strain as a source of RT for detection of RTI antibodies in cats. -KEY WORDS: feline immunodeficiency virus, reverse transcriptase assay, RT-inhibiting antibody.

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Analysis and dissociation of anti‐HIV effects of shRNA to CCR5 and the fusion inhibitor C46

Ledger

Howe

Turville

et al. 2018

The Journal of Gene Medicine

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Poly A-linked colorimetric microtiter plate assay for HIV reverse transcriptase

Cited by 85 publications

References 11 publications

Measurement of Reverse Transcriptase of Feline Immunodeficiency Virus by Poly A-Linked Colorimetric Assay.

Measurement of Reverse Transcriptase of Feline Immunodeficiency Virus by Poly A-Linked Colorimetric Assay.

Quantitative Measurement of Antibody Inhibiting Reverse Transcriptase Activity in Cats Naturally Infected with Feline Immunodeficiency Virus.

Analysis and dissociation of anti‐HIV effects of shRNA to CCR5 and the fusion inhibitor C46

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