Comparable sensitivities for detection of HIV-1 reverse transcriptase (RT) and other polymerases by RT assays requiring no radioisotopic materials

ELSEVIER

Journal of Virological Methods Journal of Virological

Methods 53 (1995) 235-244

Comparable sensitivities for detection of HIV-l reverse transcriptase ( RT) and other polymerases by RT assays requiring no radioisotopic materials Kouichi Sano a,*, Fumitorno Odawara a,b,Takashi Nakano a, Shinichi Morimatsu a, Tsumukata Nakamura a, Yukiko Saitoh a, Yan Jiang a,1,Hideo Misaki b, Yutaka Sakai a, Masuyo Nakai a aDepartment of Microbiology,

Osaka Medical College, 2-7 Daigaku-machi, Takatuki-shi, Osaka 569, Japan b Diagnostic Division, Asahi Chemical Industry Co. Ltd., 632-I Mifuku, Shizuoka 410-23, Japan Accepted

1 March 1995

Abstract An improved non-radioisotopic (Non-RI) reverse transcriptase (RT) assay with a templateprimer-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-l) were assessed. Two HIV-l 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-l substance. Keywords:

Reverse transcriptase;

RT assay; Non-RI; HIV-1 culture;biotin-dUTP

* Corresponding author. Fax: +81 (726) 84 6517. ’ Present address: Chinese PLA Academy of Military Medical Science, People’s Republic 0166-0934/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166.0934(95)00028-3

of China.

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1. Introduction The production of human immunodeficiency virus (HIV) in culture (Feorino et al., 1987; Burger et al., 1988; Lee et al., 1988; Land et al., 1989; Gupta et al., 1992; Clarke et al., 1993) and the development of an anti-reverse transcriptional inhibitor (Gronowitz et al., 1992) have become necessary. Reverse transcriptase (RT) assay is an important method not only for the study of HIV, but also for identifying new retroviruses (Gupta et al., 1992). An RT assay which does not requiring radioisotopic materials has been developed and rapidly improved in recent years (Lee et al., 1990; Sano et al., 1990; Porstmann et al., 1991; Cook et al., 1992; Eberle and Seibl, 1992; Greene et al., 1992; Urabe et al., 1992; Hammer et al., 1993; Nocito et al., 1993; Suzuki et al., 1993; Nakano et al., 1994) in response to the requirement of biosafety and radiosafety (Stinson et al., 1990). An improved version has been described (Nakano et al., 1994) of the non-radioisotopic (Non-RI) RT assay developed by Urabe et al. (1992). This assay uses a template-primer-immobilized microtiter plate and biotinylated deoxyuridine-triphosphate (BIO-dUTP). We simplified and improved the sensitivity of the assay (Nakano et al., 1994). Recently ‘RT assay, non-radioactiveTM ’ (Non-RA RT assay) has become commercially available for the detection of pelleted viruses and purified RTs. It uses BIO-dUTP to capture DNA produced by streptavidin @A)-immobilized microtiter plates and digoxigenin dUTP (DIG-dUTP) for labeling DNA (Eberle and Seibl, 1992). Comparison of these two assays indicated that the Non-RI RT assay is superior to the Non-RA RT assay in sensitivity and simplicity.

2. Materials

and methods

strain (Barre-Sinoussi et al., 1983) was grown in Molt-4 cells The HIV-l LAV-l,,u and the culture fluid was harvested. The fluid was centrifuged at 250 g for 10 min and the supernatant was filtered through a Milex HA filter. A portion of the filtrant served as the virus in the culture supernatant and another portion was ultracentrifuged at 40,000 g for 60 min at 4-10°C. The ultracentrifuged pellet was solubilized in 0.5% Triton X-100 and used as the pelleted virus sample. The pelleted virus and virus in the culture supernatant were diluted with 0.5% Triton X-100 and RPMI-1640 medium containing 10% fetal bovine serum, respectively, and mixed with the same volume of 2X lysis buffer containing 80 mM KCl, 2.5 mM DTT, 0.75 mM EDTA, 0.5% Triton X-100 and 50 mM Tris-HCl (pH 7.8). Three purified polymerases, Rous-associated virus-2 RT (RAV-2 RT), avian myeloblastosis virus RT (AMV RT) and Escherichia coli DNA polymerase I, were purchased from Takara Co., and recombinant HIV-1 RT (rHIV-1 RT) from Seikagaku Co. Enzyme samples were diluted with lysis buffer from the Non-RA RT assay kit in X2 serial dilutions. In some experiments, 20 and 200 mu/ml of rHIV-1 RTs in the lysis buffer were mixed with the same volume of RPMI-1640 medium, uninfected Molt-4 cell culture supernatant, or 0.2 mg/l of biotin solution in 50 mM Tris-HCl buffer (pH 7.8). All experiments were carried out in duplicate and the results expressed as the mean absorbance. The corresponding diluent was measured in quadruplicate, and the mean plus three times S.D. was determined as the cutoff value in

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the sensitivity study. The Non-RI RT assay system consisted of: an aminated microtiter plate (Sumitomo BakeLite Inc.) to which a poly(rA)-oligo(dT) was bound; 2X RT reaction mixture consisting of 6 PM biotin-11-deoxyuridine triphosphate (BIO-dUTP), 235 PM deoxythymidine triphosphate (dTTP), 18 mM MgCl,, 0.9 mM dithiothreitol (DIT), 2.16 mM glutathione (reduced), 0.36% Triton X-100, 3.6% ethyleneglycol, 252 mM KC1 and 0.02% NaN, in 180 mM HEPES buffer (pH 7.8); 83 ng/ml of alkaline phosphatase (ALP)-labeled streptavidin (SA) in 50 mM Tris-HCl buffer (pH 7.5); ALP substrate solution containing 1 mg/ml paranitrophenylphosphate (PNPP) and 1 mM MgCl, in 0.5 M diethanolamine-HCl buffer (pH 9.5); RT stopping solution (5 M NaCl); 1 N NaOH for ALP stopping solution; and washing buffer containing 0.02% Tween 20, 0.1 M NaCl, 0.05 M MgCl, in 0.05 M Tris-HCl buffer (pH 7.5). A fifty-microliter portion of the sample was mixed with the same volume of 2X reaction mixture in a template-primer-immobilized well and incubated at 37°C for 1, 3 or 15 h. The RT reaction was terminated by adding 10 ~1 of RT stopping solution, and the mixture was placed at room temperature for 5 min. The well was washed with washing buffer; 100 ~1 of SA-ALP solution was added followed by incubation at 37°C for 60 min. The well was washed again with washing buffer. After washing, 150 ~1 of ALP substrate solution was added and the system was incubated at 37°C for 30 min prior to the addition of 50 ~1 of ALP stopping solution. Absorbance was measured on a microplate reader (NJ 2001, Nippon InterMed Inc.) at a wavelength of 405 nm. A Non-RA RT assay kit (Boehringer Mannheim Inc.) was purchased from Nakarai Tesque (Osaka, Japan), and consisted principally of: an SA-immobilized microtiter plate; reaction mixture containing 319 mM KCl, 33 mM MgCl,, 11 mM DTI, digoxigenin-dUTP (DIG-dUTP), BIO-dUTP, dTTP and poly(rA)-oligo(dT) in 50 mM Tris buffer (pH 7.8); 10 U peroxidase (POD&labeled anti-DIG antibody solution (anti-DIG-POD); POD substrate solution containing 1 mg/ml of ABTS; and washing buffer (pH 7.0). The chemicals were reconstituted, diluted and mixed, and used according to the attached instructions. In brief, 40 ~1 of sample were mixed with 20 ~1 reaction buffer in a U-bottom microtiter plate and placed at 37°C for 1, 3 or 15 h. The mixture was transferred to an SA-fixed microtiter well and incubated at 37°C for 60 min for capture of synthesized DNA through a BIO-SA complex onto the well. After washing the well, 200 ~1 anti-DIG-POD solution was added and the system was incubated at 37°C for 60 min before the next washing. Colorization was conducted with POD substrate solution, and absorbance was measured according to the instructions. Radioisotopic (RI) RT assay was performed as described by Lee et al. (1987) with 10 &i of [3H]dTTP and 3-hour incubation. We measured the HIV-l virion with HIV-l p24 antigen capture enzyme immunoassay (AC-EIA; Abbott Laboratories, Japan) according to the instructions. To determine whether this assay could be used to clarify the mechanism of the RT inhibitor, Polygonum caspidatum extract was used. The extract was prepared as described elsewhere (Jiang et al., 1994). 3. Results The sensitivity of each of the two assay systems for detecting various purified polymerases is shown in Table 1. The Non-RI RT assay was more sensitive than the

238 Table 1 Detection

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of

Vimlogical

Methods

53 (1995) 235-244

of various polymerases

Polymerases

(mu/ml)

RAV-2 RT 200 20 2 0.2 0.02 0.002 0 AM RT 200 20 2 0.2 0.02 0.002 0

(positive/negative)

Non-RA *

Non-RI

>2 (+) 0.261 0.111 0.099 0.098 0.098 0.098

>2 >2

(+) (+) 1.767 ( + ) 0.405 ( + ) 0.187 (-) 0.154 (-) 0.113(-)

>2 >2 >2

(+) (+) (+) 0.581 (+ ) 0.192 (-) 0.143 (-1 0.154(-j

>2

(+) (-) (~) (- ) (- )

( -)

(+) 0.220 C-j 0.106(-) 0.093 ( - ) 0.094 (-

)

0.095 ( - ) 0.173 (-)

E. coli polymerase 200 20 2 0.2 0.02 0.002 0 Recombinant 200 20 2 0.2 0.02 0.002 0

Absorbance,,,;

I >2 >2

(+) (+) 0.921 0.284 0.116 0.100 0.173

(+ ) (+ ) (-) (- ) (-)

>2 (+) >2 (+) >2 (+) >2 (+) 0.694 (+) 0.234 (+) 0.154 (-)

HIV-1 RT >2 >2

(+) (+) 1.664(+) 0.272 (+) 0.118 (-) 0.099 (- 1 0.132 (-)

>2 >2 >2

(+) (+I (+I 1.203 0.338 0.165 0.149

(+) (+) (-) (-)

Cutoff b 0.259

0.234

The RT reaction was carried out under incubation at 37°C for 15 h. Enzymes were diluted with lysis buffer from the Non-RA RT assay kit. a Measurement of RT activity was performed in the enhanced calorimetry mode provided with the kit. In this mode, the negative control showed high levels of background. b Cutoff value = mean + 3 X SD. of absorbance of the corresponding diluent.

Non-RA RT assay for all samples. In measurement of polymerase from AMV, RAV-2 and E. coli, the Non-RI RT assay was, respectively, one thousand times and one hundred times more sensitive than the Non-RA RT assay in end-point of dilution. For rHIV-1 RT, the Non-M RT assay was one-tenth as sensitive as the Non-RI RT assay.

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The sensitivities of these two RT assays for the detection of RTs from virus particles, and sensitivities of sensitive assays, sensitive RI RT (Lee et al., 1987) and commercially available AC-EIA assays are shown in Table 2. The incubation periods for RT reactions were identical to those for the immunoreaction of HIV-l p24 AC-EIA. Detection of RT from the pelleted virus in Non-RI RT assay was 16 times more sensitive than Non-RA RT assay. The sensitivity of AC-EIA for measuring the pelleted virus could not be determined due to the unclear end-point. For measurement of RT from culture supernatant, Non-RI RT assay was 64 times more sensitive than Non-RA RT assay, but 16 times less sensitive than AC-EIA for detection of the virus. To determine the reasons for differences in sensitivity for the pelleted and clarified viruses in Non-RA RT assay, a detectable amount of rHIV-1 was mixed with fresh and culture media, and with biotin solution. The results are shown in Table 3. In the Non-RA RT assay, absorbance decreased remarkably by mixing with the media and biotin solution; decrease in the Non-RI RT assay was much less. The extract of Polygonum caspidatum in the assay was also examined. The extract completely inhibited the binding of RT to the template-primer immobilized on the microplate, partially inhibited polymerization, and blocked the template-primer at a dilution of 1 : 50 (Fig. 1). At dilutions of 1: 500, 1 : 5000 and 1 : 50,000, only binding inhibition was observed (Fig. 1).

4. Discussion The current Non-RI RT assay uses a primer immobilized microtiter plate and biotinylated dUTP (Urabe et al., 1992). Since serum inhibited template hybridization to the primer in this assay, the sensitivity for measuring culture fluid decreased. The sensitivity was improved by hybridization of the template to the primer prior to RT reaction (Nakano et al., 1994). The sensitivities of Non-RA RT and the latter Non-RI RT assays were compared. The sensitivities of both assays differed, depending on the polymerase including E. coli Pol-I possessing both DNA- and RNA-dependent DNA polymerase activity (Wells et al., 1972), possibly due to differences in enzyme features in polymerization. For example, one polymerase may readily incorporate DIG-dUTP and BIO-dUTP, while others may not. The Non-RA RT assay, based on the previous report (Eberle and Seibl, 1992), appears less sensitive than the Non-RI RT assay for measuring purified polymerase. For measurement of pelleted HIV-l and rHIV-1 RTs, the Non-RA assay was lo-16 times less sensitive than the Non-RI RT assay. The Non-RA RT assay, however, showed remarkably less sensitivity when RT from HIV-l in culture supernatant was measured. Eberle and Seibl(l992) indicated free BIO-dUTP to compete with incorporated BIO-dUTP in the newly synthesized DNA in the Non-RA RT assay, since this assay uses BIO-dUTP to capture DNA by SA. The culture medium includes D-biotin as a vitamin; RPMI-1640 culture medium, preferred for culturing HIV-l-infected lymphocytes, includes 2 mg/l of D-biotin. Absorbance from detectable amounts of rHIV-1 RT decreased by mixing with media, cultured media and biotin solution in the Non-RA RT assay, but not in the Non-RI RT assay. A correlation was found between decrease of absorbance and D-biotin concentration (data not shown). In the Non-RI RT

K. Sano et al. /Journal

240 Table 2 Detection

of RT from HIV-l

Virus reciprocal

particles

of dilution

Pelleted HIV-l 16 32 64 128 256 512 1024 2048 4096 8192 16384 32768 65536 131072 262144 524288 Buffer b Cutoff c HIV-1 suspension 4 8 16 32 64 128 256 512 1024 2048 4096 8192 16384 32768 65536 Buffer b Cutoff c

of Virological Methods 53 (1995) 235-244

and of polymerases

RT reactions and immunoreaction a N.T., not tested. b Corresponding diluents without ’ Cutoff values for Non-RA and corresponding diluent. In HIV-1 (Abbott, USA). Cutoff value for

medium

Absorbance,,,

cpm

Non-RA

Non-RI

AC-EL4

RI

>2

>2

(+) >2 (+I >2 (+I >2 (+)

12 >2 >2 >2

(+) (+I (+) (+) >2 (+I >2 (+) >2 (+)

194,797 119,148 82,959 43,809 23,064 15,250 N.T. N.T. N.T. N.T. N.T. N.T. N.T. N.T. N.T. N.T. 1,758 10.000

(+) 1.881 (+I 0.895 (+ ) 0.649 (+ ) 0.320 (+ I 0.163 (+I 0.111 (+I 0.060(-) 0.060 ( - ) N.T. a N.T. N.T. N.T. N.T. N.T. N.T. 0.062 ( -I 0.063 in RPMI-1640

in RPMI-1640

medium 0.202(+) 0.114 (+) 0.076 ( + ) 0.065 (-I 0.062 (-I 0.060 ( - ) 0.061 (-) 0.060 (- ) 0.060 ( - ) N.T. N.T. N.T. N.T. N.T. N.T. 0.067 ( -1 0.063

>2 >2

(+I (+) 1.637 ( + I 1.225(+) 0.570 ( +I 0.352 ( + I 0.262 (+) 0.194 (-I 0.210 (-1 0.209 (-I 0.184 (-I 0.181 (-I 0.188 (-I 0.231

>2 >2 >2 >2

(+I (+I (+I (+I 1.533 (+ I 0.936 ( + I 0.581 (+I 0.354 (+I 0.260 (+ I 0.192 (-1 0.162 (-1 0.157 (-1 0.150 (-1 0.156 (-1 0.150 (-1 0.179(-I 0.247

for EIA were conducted

1.217 (+I 0.690 (+ 1 0.418 (+I 0.237 (+ I 0.139(+) 0.085 (+) 0.053 ( -1 0.083 (+) 0.024 ( - ) 0.022 ( - ) 0.072

>2

(+I

>2 (+I >2 (+I >2 >2 >2 >2

under incubation

(+I (+I (+I (+I 1.466 0.915 0.539 0.320 0.170 0.098 0.062 0.046 0.022 0.072

(+I (+I (+ 1 (+I (+I ( +) ( -) (- ) (- >

162,466 91,774 49,134 15,194 8,690 5,444 3,508 N.T. N.T. N.T. N.T. N.T. N.T. N.T. N.T. 1,655 10,000

at 37°C for 3 h.

enzyme were measured. Non-RI assay were means of triplicate samples 3 X S.D. of absorbance of the p24 AC-E& the cutoff value was obtained from Quantum II Module A RI RT assay is discussed elsewhere (Urabe, et al., 1992).

assay, free biotin and free BIO-dUTP are washed out prior to the addition of ALP-labeled SA. There is thus no competition between free biotin and BIO-dUTP incorporated in DNA produced against SA. Biotin is removed from the culture fluid by ultracentrifuga-

K. &no et al. /Journal Table 3 Inhibitory

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effects of biotin, fresh medium and cultured medium

Amount of RT/ name of substance

Absorbance,,

a

Non-RA

Non-RI

200 mu/ml HIV-1 RT 0.2 mg/l of biotin b Fresh RPMI-1640 medium ’ Culture medium 50 mM Tris buffer (pH 7.8)

0.518 0.491 0.587 2.331

2.474 2.065 1.507 2.500

20 mU/ml HIV-1 RT 0.2 mg/l of biotin b Fresh RPMI-1640 medium ’ Culture medium 50 mM Tris buffer(pH 7.8)

0.062 0.035 0.084 0.249

1.589 1.101 1.205 1.533

a RT reaction by subtracting b Concentration ’ Molt-4 cells days. Culture

with HIV-l RT was conducted under incubation at 37°C for 1 h. The absorbance was obtained that for the corresponding diluent. of biotin was identical to that in fresh RPMI-1640 medium. were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C for 4 supernatant was harvested from the culture, centrifuged, filtered, and used as a culture medium.

tion, PEG precipitation or SA-microbead precipitation, when measuring culture fluid in the Non-RA RT assay system. This is disadvantageous, since pelleting procedures such as ultracentrifugation are tedious and biohazardous. The other commercially available RT assay kit (RT-DETECT TM; Greene et al. 1992) from DuPont, and the calorimetric RT assay (Suzuki et al., 1993) uses a biotinylated capture probe in newly synthesized DNA. Decreased sensitivity can be predicted for these systems when measuring HIV-l RT in culture fluid since they use a biotinylated probe to capture DNA onto immobilized SA in a microtiter well. Porstmann et al. (1991) reported an RT assay system using a bromodeoxyuridine triphosphate (BrdUTP) and anti-bromodeoxyuridine (anti-BrdU) antibody system. They noted that free BrdUTP inhibits the binding of antibodies to DNA (poly BrdU). Capture of a newly synthesized DNA may be inhibited by an unknown substance that cross-reacts with the anti-BrdU antibody. Other non-radioisotopic RT assay systems (Lee et al., 1990; Sano et al., 1990; Cook et al., 1992; Hammerschmid et al., 1992) all use membrane to collect the DNA product. Since the resulting membrane can be preserved, these methods are useful. It was found, however, that the harvest of DNA product on membrane is not practicable for a large number of samples, and colorization of the membrane must be carefully conducted to avoid the considerable background effect. Other methods used universally for HIV detection are the RI RT assay and HIV-l p24 AC-EIA. The Non-RI RT assay may be used in place of the sensitive RI RT assay (Lee et al., 1987) for HIV-l detection as discussed elsewhere (Nakano et al., 1994). HIV-l p24 AC-EIA is better than Non-RI RT assay in sensitivity, but not in quantitation, as shown in Table 2, since dilution end-points in AC-EIA cannot always be detected. Recently an HIV RT-typing assay was described with type-specific RT inhibiting antibodies by the use of the Non-RI RT assay, and the assay successfully

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DilUtiOIl Fig. 1. Determination of the inhibitory mechanism of the extract from Polygonurn caspidatum. The extract from dried P. caspidatum was prepared in boiled water and diluted in phosphate-buffcrcd saline as previously described (Jiang et al., 1994). The procedure used was as follows: (1) the diluted extract was mixed with the same volume of 0.5 mU 0.1% Triton X-100 solution containing 0.5 mU rHIV-1 RT, transferred to the template-primer-immobilized microplate and incubated at 37°C for 60 min prior to washing (binding inhibition); (2) enzyme solution containing 0.5 mU rHIV-1 RT in 0.1% Triton X-100 was allowed to continue binding at 37°C for 60 min to the plate and washed 5 times with washing buffer. The extract was then added to the plate and incubated at 37°C for 60 min prior to washing (polymerizing inhibition); and (3) the plate was treated with the extract at 37°C for 60 min and washed 5 times with washing buffer (template-primer blocking). The enzyme solution containing 0.5 mU rHIV-1 RT was allowed to bind to the template primer at 37°C at 60 min prior to washing. After washing, the reaction mixture was added to the well, incubated at 37°C for 60 min, and measured for bound polymerase activity.

determine the type of some clinical HIV isolates and HIV-l mutants (Urabe et al., 1994). Commercially available HIV-l p24 AC-EIA gives positive results for HIV-2, and this cannot be used for HIV-typing. AC-EIA is excellent for measuring the antigen which reacts or crossreacts to anti-HIV-l p24 antibody. AC-EIA monitors virus prodnction, but does not determine the inhibitory mechanism of anti-RT substances. We can differentiate binding inhibition from polymerizing inhibition in the Non-RI RT assay system (Fig. 1) as also shown by Gronowitz et al. (1992).

Acknowledgements This work was supported, in part, by a grant for AIDS Research from The Osaka Association for Public Health. We thank the members of our laboratory for useful help

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and encouragement. valuable comments

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We also thank Dr. Noda, A. from Takara Shuzo Co., Ltd., for his on RAV-2 RT and E. coli Pol-I.

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