Journal of Virological Metho&,
36 (1992) 171-180 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/%05.00
171
VIRMET 01283
A rapid and quantitative solution hybridization method for detection of HBV DNA in serum T. Jalava’,
M. Ranki’,
‘Orion Corporation, and ‘Department
M. Bengtstriim’,
P. Pohjanpelto2
and A. Kallio’
Orion Pharmaceutics, Biotechnology, Helsinki, Finland, of Virology, University of Helsinki, Helsinki, Finland
(Accepted 23 September
1991)
Summary A sensitive and convenient solution hybridization technique was adapted for the semi uantitative detection of hepatitis B virus DNA in serum. The assay utilizes 39S-isotope as label and biotin-avidin interaction for collection of the hybrids onto microtitre plate wells. Results are obtained as numerical values, which allow quantification. lo6 molecules of HBV DNA/ml serum could be detected by a 3-h hybridization followed by a 2-h collection reaction. By analyzing 500 l.d of serum, 30 (88%) of 34 patient sera positive for HBeAg were also positive for HBV DNA and 17 HBsAg-positive sera, 16 of which were anti-HBe-positive, were DNA-negative. The amount of HBV DNA varied from 5 x IO6 to 3 x lo9 molecules/ml. The solution hybridization method which was developed allows fast and accurate quantification of HBV DNA in serum providing an estimate of the virus titre. Hepatitis B virus; Solution hybridization;
Quantification
of DNA; Diagnostics
Introduction Hepatitis B virus (HBV) DNA in patient serum allows quantification of virus particles. It is therefore a more informative marker of active viral replication than viral proteins such as HBsAg, HBeAg, HBcAg or the HBVspecific DNA-polymerase activity (Berninger et al., 1982; Berris et al., 1987; Bonino et al., 1981; Brechot et al., 1981; Liberman et al., 1983; Scotto et al., Correspondence to: T. Jalava, Orion Corporation, Orion Pharmaceutics, Biotechnology, Valimotie 7, SF00380 Helsinki, Finland.
172
1983). Detection of HBV DNA is mainly used for monitoring the course of chronic hepatitis (Bonino et al., 198 1) and the efficacy of antiviral therapy (Peril10 et al., 1990). A variety of DNA hybridization techniques has been used for detection of HBV DNA. Most of the conventional methods are based on ‘slot’ (Liberman et al., 1983; Scotto et al., 1983) or ‘dot’ (Gerritzen and Scholt, 1990; Keijan and Bowden, 1991; Valentine-Thon et al., 1990) filter hybridizations which often require tedious sample preparation, long hybridization and autoradiographic exposure times. We have previously described a rapid solution hybridization followed by affinity-based hybrid capture technique (Ranki et al., 1983; Syvanen et al., 1986) suitable for detection of various viruses (Jalava et al., 1990; Keller et al., 1990; Urdea et al., 1987; Virtanen et al., 1983; Virtanen et al., 1984) and bacteria from a multitude of specimen materials (Kolberg et al., 1989; Palva, 1983; Palva et al., 1984). In this study, we describe the characteristics and standardization of this technique for the detection of HBV DNA in serum. Hybridization is in solution and utilizes 3sS-label in hybrid collection. The detection and microtitre plates for affinity-based analysis of HBV DNA in HBeAg-positive patient sera by this method is described.
Materials and Methods DNA reagents
The sandwich hybridization probes were derived from subtype ayw of HBV (Bichko et al., 1985). The genome of this strain was cut by BarnHI into two fragments, a 1.5kb fragment containing the core antigen gene and a 1.7-kb fragment containing the surface antigen gene. The 1.5-kb fragment was cloned into bacteriophage Ml3 mp 10 and 11 and was used as the capture probe. The 1.7-kb fragment was cloned into pBR 322 and was used as the detector probe. The HBV DNA cloned in pBR 322 was used as a positive control in hybridizations. The detector probe was labelled by nick-translation (Rigby et al., 1977) using [3sS]dCTP (Amersham, U.K.) and purified using a Bio-gel P30 column followed by phenol extraction The specific activity of the probe was 2.5 x 10’ cpm ug-’ DNA. The capture probes were chemically biotinylated (Bengstsriim and Jungell-Nortamo, to be published). The hybridization
reaction
The probe, control HBV DNA and pretreated serum samples were denatured at 100°C for 5 min, whereafter the hybridizations were carried out in eppendorf tubes in a final volume of 110 ul at 65°C for 3 h. The reaction mixture contained 0.66 M NaCl, 65 mM sodium-citrate, 0.3 mM EDTA, 0.1 M Pod-buffer (pH 6.6), 0.02% Ficoll, 0.2% polyvinylpyrrolidone, 0.5%
173
polyethyleneglycol (4000), 1.2 mg/ml BSA, 0.3% SDS and the denatured and neutralized target DNA, 500000 cpm, (about 3 x lo* molecules) of denatured detector probe DNA and the biotinylated capture DNAs (about 6 x lo9 molecules). The control HBV DNA was diluted in PBS if not otherwise indicated. Collection and measurement
of the hybrids
After hybridization the hybrids were collected onto microtitre plates coated with streptavidin (Orion Diagnostica, Finland). The collection was for 2 h at 37°C using a microtiter plate shaker. After collection the plates were washed with 200 pl of prewarmed (+ 55°C) washing solution for 2 min six times (Jalava et al., 1990). The captured hybrids were eluted twice with 150 ~1 of 0.2 M NaOH and the radioactivity was quantified by counting in a scintillation counter for 5 min. Interpretation of the results was done according to the standard curve measured simultaneously with patient sera. The results obtained by control HBV DNA as a target are given as means of five parallel determinations. Serum samples A serum pool from healthy blood donors to be used as a negative control was obtained from the Finnish Red Cross. 51 HBsAg-positive patient sera of which 34 were HBeAg- and 16 anti-HBe-positive and one negative for both HBeAg and anti-HBe and 70 patient sera negative for HBsAg were obtained from the Department of Virology, University of Helsinki. HBsAg was determined using automated IMx system (Abbott) and HBeAg and anti-HBe were determined using enzyme immuno-assay (Abbott). These sera were a random collection of samples accumulated by the Department of Virology, University of Helsinki between March 1989 and December 1990 and they were stored frozen until tested. Serum pretreatment For 500 ~1 specimen volume the following pretreatment was designed: first it was incubated in protease solution (100 pg/ml protease, 2.5% SDS, 10 mM Tris-HCI, pH 8.8, 0.24 mg/ml HS-DNA) for 2 h at 55°C. The undigested proteins were precipitated by ammonium acetate at room temperature, whereafter the DNA in the supernatant was precipitated with ethanol (Maniatis et al., 1989). For small serum volumes (5-50 pl) precipitation of proteins was omitted. In some experiments the DNA precipitation was done with isopropanol without removing proteins (Maniatis et al., 1982). The DNA pellet was resuspended in 30 ~1 of PBS and stored at -20°C until tested. When less than 500 ~.tlof serum was analyzed the volume was adjusted to 500 pl by adding HZ0 before the protease treatment.
174
Results Sensitivity
and specificity of the assay
The assay for detection of HBV DNA described here is based on sandwich hybridization in solution followed by affinity-based hybrid collection (Jalava et al., 1990; Syvanen et al., 1986). Target DNA is allowed to hybridize with radioactively (35S) labelled probe and a biotinylated capture DNA and the formed hybrids are subsequently collected onto microtiter wells coated with streptavidin (Fig. 1). The standard curves obtained for control HBV DNA with or without HBV DNA negative serum are shown in Fig. 2. The standard curves were similar, indicating that the DNA recovery after pretreatment was quantitative and no further purification of DNA from the serum specimen was required. There is a linear relationship between the log of the amount of target DNA and the log of the radioactivity measured from the collected hybrids. The sensitivity of the method is 5 x 10’ molecules of HBV DNA in 500 ~1of serum constantly giving a signal at least twice over the background level. The mean cpm value obtained with 5 x 10’ molecules of HBV DNA was chosen as the cut off value of the test. The effect of, serum on the hybridization reaction was tested. Varyinq amounts of HBV DNA negative control serum (5-30 ~1) was added to 5 x 10 and 5 x lo7 molecules of control HBV DNA and the specimens were subjected
AFFINITYMATRIX
Fig. 1. The hybrid
formed
in the solution
BIOTINYLATED CAPTURE
hybridization assay affinity matrix.
LABELED PROBE
after collection
onto
streptavidin
coated
175
100
Al”‘ll
"I
IO6
0
,/I,,.,
10'
I/#,
lo8
Number of target DNA
,,
‘9
7
10
molecules
Fig. 2. The standard curve obtained for control HBV DNA (A) and for HBV DNA mixed with 500 ~1 of HBV negative serum ( x) using the solution hybridization and the affinity-based hybrid collection method. lo6 molecules correspond to 3.5 pg of fully double-stranded HBV DNA.
to hybridization with or without pretreatment procedure. The untreated serum caused a concentration-dependent inhibition (2&70%) in the signal, whereas pretreated serum had no inhibitory effect when compared with results obtained without serum addition (data not shown). The specificity of the assay was confirmed by testing sera positive for a variety of viruses. No signal over the background level was recorded from 500 l.d of sera obtained from hepatitis C virus, human cytomegalovirus, EpsteinBarr virus or human immunodeticiency virus antibody-positive individuals. Kinetics of the hybridization
reaction
Fig. 3 illustrates the rate of the solution hybridization reaction for 5 x lo5 and 5 x lo7 molecules of control HBV DNA target and for one HBV DNA positive patient serum. The reaction reached its maximum in 4 h with 5 x lo7 molecules of HBV DNA and with the patient sample. The hybridization with 5 x lo5 molecules of HBV DNA was completed in 24 h. Depending on the target concentration, 42-82% of the reaction was completed in 3 h at 65°C. The background levels increased when hybridization was prolonged up to 24 h (from 6 cpm to 35 cpm). Quantzjkation
of HBV DNA from patient sera
In order to confirm the possibility of quantification by this method, 500 ~1 of a HBV DNA-positive serum was analyzed and the number of the HBV DNA molecules in serum was obtained from the standard curve. From the same serum 250-, loo-, and 50-~1 aliquots were tested. Before pretreatment the volumes of sera were adjusted to 500 ~1 by adding HBV DNA-negative serum
176
0123456
o/n Hours
Fig. 3. Kinetics of the hybridization reaction. 5 x lo7 (0) and 5 x 10’ ( x ) molecule of control HBV DNA and a pretreated HBV DNA positive patient serum (0) were allowed to hybridize with biotinylated capture DNA and 35S-labelled probe DNA for different time periods at 65°C. The signal after background subtraction (4-35 cpm) are shown.
to them. The signals obtained in response to the dilutions were identical to those of the standard curve (see Fig. 4). 17 HBeAg-positive patient sera were obtained for HBV DNA analysis. 50 ~1 and 500 ~1 of the specimen were pretreated and analyzed. The signals obtained were converted into number of HBV DNA molecules using the standard curve. The results are summarized in Table 1. Sixteen of the samples were positive for HBV DNA. In nine of the 17 samples (l-9) the cpm values obtained with 500 I.LI
1000
7.
I
b
Number of target DNA molecules Fig. 4. Quantification of HBV DNA from a clinical specimen as correlated with serum dilution. Dilutions of pretreated HBV DNA-positive serum (0) and control DNA (0) were subjected to the hybridization test and the signals obtained are presented.
TABLE 1 Amount of HBV DNA in HBeAg-positive Patient number
Solution hybridization
patient sera
with serum
500 p1
1 2 3 4 5 6 : 9 10 11 12 13 14 1.5 16 17
50 ul
Signal (cpm)
HBV DNA” molecules/ml
Signal (cpm)
HBV DNA” molecules/ml
17501 17438 15489 13924 12235 10051 9522 8884 8091 7109 6800 3950 2449 1357 1000 513 23
>10* >10* >10* >10* >10* >10* >10* >10* >10* 1.0 x 9.2 x 5.2 x 3.1 x 1.6 x 1.2 x 6.4 x neg.
3249 4359 5298 10311 2241 3065 1794 2361 2502 4214 969 617 388 140 118 61 16
4.2 x 5.7 x 7.0 x >10* 2.6 x 3.8 x 2.2 x 2.9 x 3.2 x 5.5 x 1.1 x 6.8 x 4.2 x 1.3 x 1.1 x neg. neg.
lo* lo7 lo7 lo7 lo7 107 lo6
lo* 108 108 10’ lo* 108 10’ 10’ lo* 108 lo7 lo7 107 lo7
a Calculation was according to the signals obtained with different numbers of molecules of positive control which were: negative (18 cpm); 5 x lo5 (108 cpm); 5 x lo6 (894 cpm); 5 x lo7 (7146 cpm).
serum exceeded those of the standard curve. Therefore the quantity of HBV DNA was obtained from analyzing the 50 ul serum aliquot. An additional serum dilution (1:lO) was required for exact quantification of patient No. 4 HBV DNA (2.7 x lo9 molecules/ml). 34 HBsAg-positive patient sera, 17 positive for HBeAg, 16 positive for antiHBe and 1 negative for both HBeAg and anti-HBe were additionally analyzed by the assay. The results are summarized in Table 2. Thus, of 34 patients positive for HBeAg, 30 (88%) were HBV DNA-positive. All 17 HBeAgnegative sera were HBV DNA-negative by this assay. In addition, 500- and 50-ul aliquots of 70 randomly selected patient sera, negative for HBsAg, were tested by the hybridization assay in four test series. TABLE 2 Detection
of HBV DNA from seraa by solution hybridization
HBV DNA
Positive (30) Negative (21) YSera were pretreated
assay as correlated
HBeAg Positive
Negative
30 4
1;
by precipitation
with ethanol or with isopropanol.
with HBeAg
178
69 samples out of 70 were negative when 500 ul of the serum was analyzed with a mean cpm value of 22 cpm (SD + 13 cpm). The 50-ul specimen aliquots were all negative, the mean cpm value being 18 cpm (SD f 9 cpm). A new serum sample was obtained from the HBsAg-negative but HBV DNA-positive patient. In the reanalysis it remained HBV DNA-negative. The cut off in the test series was 106 cpm (SD + 10 cpm). These results show that the positive patient samples were clearly distinguished from the negative ones.
Discussion The presence of HBV in serum is conventionally determined by assay of HBeAg. However, since this antigen can exist in a free form, the correlation with infectious particles is not absolute (Gerber and Thung, 1985). Endogenous DNA polymerase may be a better indicator of intact viral particles (Hung et al., 1975; Kaplan et al., 1973; Robinson, 1975). However, because the DNA polymerase reaction is catalytic rather than stoichiometric, measurements must be interpreted with caution. The presence of DNA contained in viral particles offers a more accurate determination of viral replication and is more sensitive than the DNA polymerase assay (Bonino et al., 1981; Fagan et al., 1985; Kuhns et al., 1989; Weller et al., 1982). PCR is the most sensitive method for HBV DNA detection but it is too complicated for routine diagnostic purposes (Gerken et al., 1991). The quantification of HBV DNA in serum is important when choosing suitable antiviral therapy for the patient. It has been shown that patients with low-to-moderate levels of HBV DNA and HBeAg in serum responded to alpha interferon therapy, whereas patients with high levels of HBV DNA did not respond (Hoofnagle, 1990). Successful treatment can be defined by disappearance of HBV DNA together with HBeAg during or shortly after treatment. The sensitivity of the test described now was 5 x lo5 HBV DNA molecules, which is about 3.5 pg of HBV in 1 ml of serum. It is close to the theoretical limit for scintillation counting of 35S-labelled DNA. The sensitivity was comparable to that obtained with radioactive probes, labelled with 32P and 12’1, used in many filter and solution hybridization assays (Pontisso et al., 1989; ValentineThon et al., 1990). The test is linear over several orders of magnitude and analysis of 50-500 ul of HBV DNA-positive patient serum yielded similar HBV DNA concentrations (Table 1, Fig. 4). Although the kinetics of hybridization reaction in 3 h depended on the concentration of HBV target DNA present in the reaction (Fig. 3) the results are quantifiable because they are interpreted from the standard curve. To demonstrate the utility of the assay described in this paper, patient sera were analyzed for the presence of HBV DNA. 30 of 34 HBeAg positive samples were found to be DNA positive. The correlation of DNA positivity to the HBeAg positivity was similar to that obtained by other HBV DNA detection methods (Valentine-Thon et al., 1990; Keller et al., 1990; Hoofnagle, 1990). The
179
amount of HBV DNA in these sera varied from 5 x lo6 to 3 x lo9 molecules/ ml. One out of 70 patient sera negative for HBsAg was DNA positive. Further information on the patient’s hepatitis status was not available. From the results obtained in this study and experiments described earlier (Syvanen et al., 1986; Jalava et al., 1990) it can be concluded that the solution hybridization technique provides an easy-to-use and quantitative method for the identification of HBV DNA in serum. Based on the method presented here we developed an AffrProbe HepB test kit for identification and semiquantification of the HBV DNA in serum.
Acknowledgements
The excellent technical assistance of Raija Lahdensivu from the Department of Virology, University of Helsinki and Sini Heinonen is greatly acknowledged. We thank Dr Hans Soderlund for fruitful discussions. References Beminger, M., Hammer, M., Hoyer, B. and Gerlin, J.L. (1982) An assay for the detection of the DNA genome of hepatitis B virus in serum. J. Med. Virol. 9, 57-68. Berris, B., Sampliner, E.R., Sooknanan, R. and Feinman, S.V. (1987) Hepatitis B virus DNA in asymptomatic HBsAg carriers: comparison with HBeAg/antiHBe status. J. Med. Virol. 23,233239. Bichko, V., Pushko, P., Dreilina, D., Pumpen, P. and Gren, E. (1985) Subtype ayw variant of hepatitis B virus, DNA primary structure analysis. FEBS 185, 208-212. Bonino, F., Hoyer, B., Nelson, J., Engle, R., Verme, G. and Germ, J. (1981) Hepatitis B virus DNA in sera of HBsAg carriers: a marker of active hepatitis B virus replication in the liver. Hepatology 1,386391. Brechot, C., Hadchouel, M., Scotto, J., Degos, F., Chamay, P., Trepo, C. and Tiollais, P. (1981) Detection of hepatitis B virus DNA in liver and serum: a direct appraisal of the chronic carrier state. Lancet ii, 765768. Fagan, E., Guarner, P., Perera, S., Troebridge, R., Rolando, N., Davison, F. and Williams, R. (1985) Quantification of hepatitis B virus DNA (HBV DNA) in serum using the spot hybridization technique and scintillation counting. J. Virol. Methods 8, 199-206. Gerber, M.A. and Thung, S.N. (1985) Biology of disease: molecular and cellular pathology of hepatitis B. Lab. Invest. 52, 572-590. Gerken, G., Paterlini, P., Manns, M., Housset, C., Terre, S., Dienes, H., Hess, G., Gerlich, W., Berthelot, P., Meyer zum Buschenfelde, K. and Brechot, C. (1991) Assay of hepatitis B virus DNA by polymerase chain reaction and its relationship to pre-S- and S-encoded viral surface antigen. Hepatology 13, 158-166. Gerritzen, A. and Scholt, B. (1990) A nonradioactive riboprobe assay for the detection of hepatitis B virus DNA in human sera. J. Virol. Methods 30, 31 l-318. Hoofnagle, J.H. (1990) Chronic hepatitis B. N. Engl. J. Med. 323, 337-339. Hung, P.P., Mao, J.C.H., Ling, C.M. and Overby, L.R. (1975) Hybridization of Dane particle DNA with the free plasma DNA of hepatitis carrier. Nature 253, 571-572. Jalava, T., Kalho, A., Leinonen, A.W. and Ranki, M. (1990) A rapid solution hybridization method for detection of human papilloma viruses. Mol. Cell. Probes 4, 341-352. Kaplan, P.M., Greenman, R.L., Germ, J.L., Purcell, R.H. and Robinson, W.S. (1973) DNA polymerase associated with human hepatitis B antigen. J. Virol. 12, 995-1005. Keijan, G. and Bowden, S. (1991) Digoxigenin-labeled probes for the detection of hepatitis B virus DNA in serum. J. Clin. Microbial. 29. 506-509.
180
Keller, G.H., Huang, D., Shih, W. and Manak, M.M. (1990) Detection of hepatitis B virus DNA in serum by polymerase chain reaction ampli~~tion and microtiter sandwich hyb~di~tion. J. Clin. Microbial. 28, 141 I-1416. Kolberg, J.A., Besemer, D.J., Stempier, M.M. and Urdea, MS. (1989) The specificity of pilin DNA sequences for the detection of pathogenic Neisseria. Mol. Cell. Probes 3, 59-72. Kuhns, M.C., McNamara, A.L., Perrillo, R.P., Cabal, C.M. and Campbell, CR. (1989) Quantification of hepatitis B viral DNA by solution hybridization: comparison with DNA polymerase and hepatitis B antigen during antiviral therapy. J. Med. Virol. 27, 274-281. Liberman, H.M., LaBreque, D.R., Kew, MC., Ha~iyannis, S.J. and Shafriz, D.A. (1983) Detection of hepatitis B virus DNA directly in human serum by a simplified molecular hybridization test: comparison to HBeAg/anti-HBe status in HBsAg carriers. Hepatology 3, 285-291. Mania&, T., Fritsch, E.F. and Sambrook, J. (1989) In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, pp. ElO-El5 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, p. 401. Palva, A.M. (1983) omp A gene in the detection of Escherichia cob and other Enterobacteriaceae by nucleic acid sandwich hybridization. J. Clin. Microbial. 18, 92-100. Palva, A., Jousimies-Somer, H., Saikku, P., Vaiinlnen, P., Siiderlund, H. and Ranki, M. (1984) Detection of Chlamydia trachomatis by nucleic acid sandwich hybridization. FEMS Len. 23, 83-89. Perillo, R., Schiff, R., Davis, G., Bodenheimer, II., Lindsay, K., Payne, J., Dienstag, J., O’Brien, C., Tamburro, C., Jacobson, I., Sampliner, R., Feit, D., Le~ow~tch, J., Kuhns, M., Meschievitz, C., Sanghvi, B., Albrecht, J., Gibas, A. and the hepatitis interventional therapy group (1990) A randomized, controlled trial of interferon alpha-2b alone and after prednisone withdrawal for the treatment of chronic hepatitis B. N. Engl. J. Med. 323, 295-301. Pontisso, P,, Ruvoletto, M.G., Fattovich, G., Chemello, L., Morsica, G., Brollo, L., Matteotti, V. and Alberti, A. (1989) Serum HBV-DNA in anti-HBe positive patients detected by filter and liquid phase hyb~di~tion assays. Mol. Cell. Probes 3, 245-249. Ranki, M., Palva, A., Virtanen, M., Laaksonen, M. and Soderlund, H. (1983) Sandwich hybridization as a convenient method for the detection of nucleic acids in crude samples. Gene 21, 77-85. Rigby, P.W.J., Dieckmann, M., Rhodes, C. and Berg, P. (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase 1. J. Mol. Biol. 113,237251. Robinson, W.S. (1975) DNA and DNA polymerase in the core of the DNA particle of hepatitis B. Am. J. Med. Sci. 270, 151-159. Scotto, J., Hadchouel, M., Hery, C., Yvart, J., Tiohais, P. and Brechot, C. (1983) Detection of hepatitis B virus DNA in serum by single spot hybridization technique: comparison with results for other viral markers. Hepatology 3, 279-282. Syv&nen, C.-A., Laaksonen, M. and Siiderlund, H. (1986) Fast quantification of nucleic acid affinity based hybrid collection. Nucleic Acids Res. 14, 5037-5048. Urdea, M.S., Running, J.A., Horn, T., Clyne, J., Ku, L. and Warner, B.D. (1987) A novel method for the rapid detection of specific nucleotide sequences in crude biological samples without blotting or radioactivity: application to the analysis of hepatitis B virus in human serum. Gene 61, 253-264. Valentine-Thon, E., Steinmann, J. and Arnold, W. (1990) Evaluation of the commercially available HepProbe kit for detection of hepatitis B virus DNA in serum. J. Clin. Microbial. 28, 39-42. Virtanen, M., Laaksonen, M., Soderlund, H., Palva, A., Halonen, P. and Ranki, M. (1983) Novel test for rapid viral diagnosis: detection of adenovirus in n~opha~ngeal aspirates by means of nucleic acid sandwich hyb~~zation. Lancet i, 381-383. Virtanen, M., SyvPnen, A.-C., Oram, J., Soderlund, H. and Ranki, M. (1984) Cytomegaiovirus in urine: detection of viral DNA by sandwich hybridization J. Clin. Microbial. 20, 1083-1088. Weller, I., Fowler, M., Monjardino, J. and Thomas, H. (1982) The detection of HBV-DNA in serum by molecular hybridization: a more sensitive method for the detection of complete HBV particles. J. Med. Virol. 9, 273-280.
36 (1992) 171-180 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/%05.00
171
VIRMET 01283
A rapid and quantitative solution hybridization method for detection of HBV DNA in serum T. Jalava’,
M. Ranki’,
‘Orion Corporation, and ‘Department
M. Bengtstriim’,
P. Pohjanpelto2
and A. Kallio’
Orion Pharmaceutics, Biotechnology, Helsinki, Finland, of Virology, University of Helsinki, Helsinki, Finland
(Accepted 23 September
1991)
Summary A sensitive and convenient solution hybridization technique was adapted for the semi uantitative detection of hepatitis B virus DNA in serum. The assay utilizes 39S-isotope as label and biotin-avidin interaction for collection of the hybrids onto microtitre plate wells. Results are obtained as numerical values, which allow quantification. lo6 molecules of HBV DNA/ml serum could be detected by a 3-h hybridization followed by a 2-h collection reaction. By analyzing 500 l.d of serum, 30 (88%) of 34 patient sera positive for HBeAg were also positive for HBV DNA and 17 HBsAg-positive sera, 16 of which were anti-HBe-positive, were DNA-negative. The amount of HBV DNA varied from 5 x IO6 to 3 x lo9 molecules/ml. The solution hybridization method which was developed allows fast and accurate quantification of HBV DNA in serum providing an estimate of the virus titre. Hepatitis B virus; Solution hybridization;
Quantification
of DNA; Diagnostics
Introduction Hepatitis B virus (HBV) DNA in patient serum allows quantification of virus particles. It is therefore a more informative marker of active viral replication than viral proteins such as HBsAg, HBeAg, HBcAg or the HBVspecific DNA-polymerase activity (Berninger et al., 1982; Berris et al., 1987; Bonino et al., 1981; Brechot et al., 1981; Liberman et al., 1983; Scotto et al., Correspondence to: T. Jalava, Orion Corporation, Orion Pharmaceutics, Biotechnology, Valimotie 7, SF00380 Helsinki, Finland.
172
1983). Detection of HBV DNA is mainly used for monitoring the course of chronic hepatitis (Bonino et al., 198 1) and the efficacy of antiviral therapy (Peril10 et al., 1990). A variety of DNA hybridization techniques has been used for detection of HBV DNA. Most of the conventional methods are based on ‘slot’ (Liberman et al., 1983; Scotto et al., 1983) or ‘dot’ (Gerritzen and Scholt, 1990; Keijan and Bowden, 1991; Valentine-Thon et al., 1990) filter hybridizations which often require tedious sample preparation, long hybridization and autoradiographic exposure times. We have previously described a rapid solution hybridization followed by affinity-based hybrid capture technique (Ranki et al., 1983; Syvanen et al., 1986) suitable for detection of various viruses (Jalava et al., 1990; Keller et al., 1990; Urdea et al., 1987; Virtanen et al., 1983; Virtanen et al., 1984) and bacteria from a multitude of specimen materials (Kolberg et al., 1989; Palva, 1983; Palva et al., 1984). In this study, we describe the characteristics and standardization of this technique for the detection of HBV DNA in serum. Hybridization is in solution and utilizes 3sS-label in hybrid collection. The detection and microtitre plates for affinity-based analysis of HBV DNA in HBeAg-positive patient sera by this method is described.
Materials and Methods DNA reagents
The sandwich hybridization probes were derived from subtype ayw of HBV (Bichko et al., 1985). The genome of this strain was cut by BarnHI into two fragments, a 1.5kb fragment containing the core antigen gene and a 1.7-kb fragment containing the surface antigen gene. The 1.5-kb fragment was cloned into bacteriophage Ml3 mp 10 and 11 and was used as the capture probe. The 1.7-kb fragment was cloned into pBR 322 and was used as the detector probe. The HBV DNA cloned in pBR 322 was used as a positive control in hybridizations. The detector probe was labelled by nick-translation (Rigby et al., 1977) using [3sS]dCTP (Amersham, U.K.) and purified using a Bio-gel P30 column followed by phenol extraction The specific activity of the probe was 2.5 x 10’ cpm ug-’ DNA. The capture probes were chemically biotinylated (Bengstsriim and Jungell-Nortamo, to be published). The hybridization
reaction
The probe, control HBV DNA and pretreated serum samples were denatured at 100°C for 5 min, whereafter the hybridizations were carried out in eppendorf tubes in a final volume of 110 ul at 65°C for 3 h. The reaction mixture contained 0.66 M NaCl, 65 mM sodium-citrate, 0.3 mM EDTA, 0.1 M Pod-buffer (pH 6.6), 0.02% Ficoll, 0.2% polyvinylpyrrolidone, 0.5%
173
polyethyleneglycol (4000), 1.2 mg/ml BSA, 0.3% SDS and the denatured and neutralized target DNA, 500000 cpm, (about 3 x lo* molecules) of denatured detector probe DNA and the biotinylated capture DNAs (about 6 x lo9 molecules). The control HBV DNA was diluted in PBS if not otherwise indicated. Collection and measurement
of the hybrids
After hybridization the hybrids were collected onto microtitre plates coated with streptavidin (Orion Diagnostica, Finland). The collection was for 2 h at 37°C using a microtiter plate shaker. After collection the plates were washed with 200 pl of prewarmed (+ 55°C) washing solution for 2 min six times (Jalava et al., 1990). The captured hybrids were eluted twice with 150 ~1 of 0.2 M NaOH and the radioactivity was quantified by counting in a scintillation counter for 5 min. Interpretation of the results was done according to the standard curve measured simultaneously with patient sera. The results obtained by control HBV DNA as a target are given as means of five parallel determinations. Serum samples A serum pool from healthy blood donors to be used as a negative control was obtained from the Finnish Red Cross. 51 HBsAg-positive patient sera of which 34 were HBeAg- and 16 anti-HBe-positive and one negative for both HBeAg and anti-HBe and 70 patient sera negative for HBsAg were obtained from the Department of Virology, University of Helsinki. HBsAg was determined using automated IMx system (Abbott) and HBeAg and anti-HBe were determined using enzyme immuno-assay (Abbott). These sera were a random collection of samples accumulated by the Department of Virology, University of Helsinki between March 1989 and December 1990 and they were stored frozen until tested. Serum pretreatment For 500 ~1 specimen volume the following pretreatment was designed: first it was incubated in protease solution (100 pg/ml protease, 2.5% SDS, 10 mM Tris-HCI, pH 8.8, 0.24 mg/ml HS-DNA) for 2 h at 55°C. The undigested proteins were precipitated by ammonium acetate at room temperature, whereafter the DNA in the supernatant was precipitated with ethanol (Maniatis et al., 1989). For small serum volumes (5-50 pl) precipitation of proteins was omitted. In some experiments the DNA precipitation was done with isopropanol without removing proteins (Maniatis et al., 1982). The DNA pellet was resuspended in 30 ~1 of PBS and stored at -20°C until tested. When less than 500 ~.tlof serum was analyzed the volume was adjusted to 500 pl by adding HZ0 before the protease treatment.
174
Results Sensitivity
and specificity of the assay
The assay for detection of HBV DNA described here is based on sandwich hybridization in solution followed by affinity-based hybrid collection (Jalava et al., 1990; Syvanen et al., 1986). Target DNA is allowed to hybridize with radioactively (35S) labelled probe and a biotinylated capture DNA and the formed hybrids are subsequently collected onto microtiter wells coated with streptavidin (Fig. 1). The standard curves obtained for control HBV DNA with or without HBV DNA negative serum are shown in Fig. 2. The standard curves were similar, indicating that the DNA recovery after pretreatment was quantitative and no further purification of DNA from the serum specimen was required. There is a linear relationship between the log of the amount of target DNA and the log of the radioactivity measured from the collected hybrids. The sensitivity of the method is 5 x 10’ molecules of HBV DNA in 500 ~1of serum constantly giving a signal at least twice over the background level. The mean cpm value obtained with 5 x 10’ molecules of HBV DNA was chosen as the cut off value of the test. The effect of, serum on the hybridization reaction was tested. Varyinq amounts of HBV DNA negative control serum (5-30 ~1) was added to 5 x 10 and 5 x lo7 molecules of control HBV DNA and the specimens were subjected
AFFINITYMATRIX
Fig. 1. The hybrid
formed
in the solution
BIOTINYLATED CAPTURE
hybridization assay affinity matrix.
LABELED PROBE
after collection
onto
streptavidin
coated
175
100
Al”‘ll
"I
IO6
0
,/I,,.,
10'
I/#,
lo8
Number of target DNA
,,
‘9
7
10
molecules
Fig. 2. The standard curve obtained for control HBV DNA (A) and for HBV DNA mixed with 500 ~1 of HBV negative serum ( x) using the solution hybridization and the affinity-based hybrid collection method. lo6 molecules correspond to 3.5 pg of fully double-stranded HBV DNA.
to hybridization with or without pretreatment procedure. The untreated serum caused a concentration-dependent inhibition (2&70%) in the signal, whereas pretreated serum had no inhibitory effect when compared with results obtained without serum addition (data not shown). The specificity of the assay was confirmed by testing sera positive for a variety of viruses. No signal over the background level was recorded from 500 l.d of sera obtained from hepatitis C virus, human cytomegalovirus, EpsteinBarr virus or human immunodeticiency virus antibody-positive individuals. Kinetics of the hybridization
reaction
Fig. 3 illustrates the rate of the solution hybridization reaction for 5 x lo5 and 5 x lo7 molecules of control HBV DNA target and for one HBV DNA positive patient serum. The reaction reached its maximum in 4 h with 5 x lo7 molecules of HBV DNA and with the patient sample. The hybridization with 5 x lo5 molecules of HBV DNA was completed in 24 h. Depending on the target concentration, 42-82% of the reaction was completed in 3 h at 65°C. The background levels increased when hybridization was prolonged up to 24 h (from 6 cpm to 35 cpm). Quantzjkation
of HBV DNA from patient sera
In order to confirm the possibility of quantification by this method, 500 ~1 of a HBV DNA-positive serum was analyzed and the number of the HBV DNA molecules in serum was obtained from the standard curve. From the same serum 250-, loo-, and 50-~1 aliquots were tested. Before pretreatment the volumes of sera were adjusted to 500 ~1 by adding HBV DNA-negative serum
176
0123456
o/n Hours
Fig. 3. Kinetics of the hybridization reaction. 5 x lo7 (0) and 5 x 10’ ( x ) molecule of control HBV DNA and a pretreated HBV DNA positive patient serum (0) were allowed to hybridize with biotinylated capture DNA and 35S-labelled probe DNA for different time periods at 65°C. The signal after background subtraction (4-35 cpm) are shown.
to them. The signals obtained in response to the dilutions were identical to those of the standard curve (see Fig. 4). 17 HBeAg-positive patient sera were obtained for HBV DNA analysis. 50 ~1 and 500 ~1 of the specimen were pretreated and analyzed. The signals obtained were converted into number of HBV DNA molecules using the standard curve. The results are summarized in Table 1. Sixteen of the samples were positive for HBV DNA. In nine of the 17 samples (l-9) the cpm values obtained with 500 I.LI
1000
7.
I
b
Number of target DNA molecules Fig. 4. Quantification of HBV DNA from a clinical specimen as correlated with serum dilution. Dilutions of pretreated HBV DNA-positive serum (0) and control DNA (0) were subjected to the hybridization test and the signals obtained are presented.
TABLE 1 Amount of HBV DNA in HBeAg-positive Patient number
Solution hybridization
patient sera
with serum
500 p1
1 2 3 4 5 6 : 9 10 11 12 13 14 1.5 16 17
50 ul
Signal (cpm)
HBV DNA” molecules/ml
Signal (cpm)
HBV DNA” molecules/ml
17501 17438 15489 13924 12235 10051 9522 8884 8091 7109 6800 3950 2449 1357 1000 513 23
>10* >10* >10* >10* >10* >10* >10* >10* >10* 1.0 x 9.2 x 5.2 x 3.1 x 1.6 x 1.2 x 6.4 x neg.
3249 4359 5298 10311 2241 3065 1794 2361 2502 4214 969 617 388 140 118 61 16
4.2 x 5.7 x 7.0 x >10* 2.6 x 3.8 x 2.2 x 2.9 x 3.2 x 5.5 x 1.1 x 6.8 x 4.2 x 1.3 x 1.1 x neg. neg.
lo* lo7 lo7 lo7 lo7 107 lo6
lo* 108 108 10’ lo* 108 10’ 10’ lo* 108 lo7 lo7 107 lo7
a Calculation was according to the signals obtained with different numbers of molecules of positive control which were: negative (18 cpm); 5 x lo5 (108 cpm); 5 x lo6 (894 cpm); 5 x lo7 (7146 cpm).
serum exceeded those of the standard curve. Therefore the quantity of HBV DNA was obtained from analyzing the 50 ul serum aliquot. An additional serum dilution (1:lO) was required for exact quantification of patient No. 4 HBV DNA (2.7 x lo9 molecules/ml). 34 HBsAg-positive patient sera, 17 positive for HBeAg, 16 positive for antiHBe and 1 negative for both HBeAg and anti-HBe were additionally analyzed by the assay. The results are summarized in Table 2. Thus, of 34 patients positive for HBeAg, 30 (88%) were HBV DNA-positive. All 17 HBeAgnegative sera were HBV DNA-negative by this assay. In addition, 500- and 50-ul aliquots of 70 randomly selected patient sera, negative for HBsAg, were tested by the hybridization assay in four test series. TABLE 2 Detection
of HBV DNA from seraa by solution hybridization
HBV DNA
Positive (30) Negative (21) YSera were pretreated
assay as correlated
HBeAg Positive
Negative
30 4
1;
by precipitation
with ethanol or with isopropanol.
with HBeAg
178
69 samples out of 70 were negative when 500 ul of the serum was analyzed with a mean cpm value of 22 cpm (SD + 13 cpm). The 50-ul specimen aliquots were all negative, the mean cpm value being 18 cpm (SD f 9 cpm). A new serum sample was obtained from the HBsAg-negative but HBV DNA-positive patient. In the reanalysis it remained HBV DNA-negative. The cut off in the test series was 106 cpm (SD + 10 cpm). These results show that the positive patient samples were clearly distinguished from the negative ones.
Discussion The presence of HBV in serum is conventionally determined by assay of HBeAg. However, since this antigen can exist in a free form, the correlation with infectious particles is not absolute (Gerber and Thung, 1985). Endogenous DNA polymerase may be a better indicator of intact viral particles (Hung et al., 1975; Kaplan et al., 1973; Robinson, 1975). However, because the DNA polymerase reaction is catalytic rather than stoichiometric, measurements must be interpreted with caution. The presence of DNA contained in viral particles offers a more accurate determination of viral replication and is more sensitive than the DNA polymerase assay (Bonino et al., 1981; Fagan et al., 1985; Kuhns et al., 1989; Weller et al., 1982). PCR is the most sensitive method for HBV DNA detection but it is too complicated for routine diagnostic purposes (Gerken et al., 1991). The quantification of HBV DNA in serum is important when choosing suitable antiviral therapy for the patient. It has been shown that patients with low-to-moderate levels of HBV DNA and HBeAg in serum responded to alpha interferon therapy, whereas patients with high levels of HBV DNA did not respond (Hoofnagle, 1990). Successful treatment can be defined by disappearance of HBV DNA together with HBeAg during or shortly after treatment. The sensitivity of the test described now was 5 x lo5 HBV DNA molecules, which is about 3.5 pg of HBV in 1 ml of serum. It is close to the theoretical limit for scintillation counting of 35S-labelled DNA. The sensitivity was comparable to that obtained with radioactive probes, labelled with 32P and 12’1, used in many filter and solution hybridization assays (Pontisso et al., 1989; ValentineThon et al., 1990). The test is linear over several orders of magnitude and analysis of 50-500 ul of HBV DNA-positive patient serum yielded similar HBV DNA concentrations (Table 1, Fig. 4). Although the kinetics of hybridization reaction in 3 h depended on the concentration of HBV target DNA present in the reaction (Fig. 3) the results are quantifiable because they are interpreted from the standard curve. To demonstrate the utility of the assay described in this paper, patient sera were analyzed for the presence of HBV DNA. 30 of 34 HBeAg positive samples were found to be DNA positive. The correlation of DNA positivity to the HBeAg positivity was similar to that obtained by other HBV DNA detection methods (Valentine-Thon et al., 1990; Keller et al., 1990; Hoofnagle, 1990). The
179
amount of HBV DNA in these sera varied from 5 x lo6 to 3 x lo9 molecules/ ml. One out of 70 patient sera negative for HBsAg was DNA positive. Further information on the patient’s hepatitis status was not available. From the results obtained in this study and experiments described earlier (Syvanen et al., 1986; Jalava et al., 1990) it can be concluded that the solution hybridization technique provides an easy-to-use and quantitative method for the identification of HBV DNA in serum. Based on the method presented here we developed an AffrProbe HepB test kit for identification and semiquantification of the HBV DNA in serum.
Acknowledgements
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