A nonradioactive hybridization assay for the detection of hepatitis delta virus RNA in serum is .... recombinant HBV DNA or to 1 ng of bacteriophage lambda.
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1994, P. 1112-1114
Vol. 32, No. 4
0095-1137/94/$04.00+0 Copyright C 1994, American Society for MicrobiologY
Chemiluminescent Hybridization Assay for Hepatitis Delta Virus RNA in Serum MARGARET GALLAGHER,* TIMOTHY T. MORRIS, AND HOWARD A. FIELDS Hepatitis Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333 Received 28 July 1993/Returned for modification 27 September 1993/Accepted 24 January 1994
A nonradioactive hybridization assay for the detection of hepatitis delta virus RNA in serum is described. This assay utilizes a digoxigenin-labeled RNA hybridization probe and chemiluminescent immunodetection. The probe can detect as little as 0.4 pg of cDNA with a 60-min exposure. Results obtained are in agreement with serological indicators of hepatitis delta virus infection and are comparable to those obtained by hybridization assays employing radioactively labeled RNA.
Hepatitis delta virus (HDV) is a viroid-like human pathogen that requires hepatitis B virus (HBV) for successful maturation. HDV coinfection with HBV usually results in acute hepatitis followed by resolution of the disease. Alternatively, HBV carriers may be superinfected by HDV, which frequently results in the establishment of persistent HDV infections that frequently lead to more severe liver disease. Thus, HDV infection poses a serious public health problem in those parts of the world with high-level HBV endemism. Determination of HDV RNA levels in serum by molecular hybridization is a noninvasive method to assess viral replication and infectivity and has been used to demonstrate the presence of HDV in the sera of patients with both acute and chronic HDV infections (9). Although 32P-labeled probes provide highly sensitive detection, their use is associated with a number of problems such as safety considerations during handling and disposal, and their short half-life necessitates frequent probe synthesis. An alternative nonradioactive labeling and detection system which employs the steroid hapten digoxigenin has recently been developed. Hybridized probes are detected by an enzyme-linked immunoassay using polyclonal anti-digoxigenin Fab fragments conjugated to alkaline phosphatase. This system has been reported to have a sensitivity comparable to that obtained with the same probes labeled with 32p (3). An additional advantage of this system is the ability to obtain high yields of digoxigenin-labeled RNA by in vitro transcription (6). Recently, several investigators have demonstrated that chemiluminescent enzyme substrates enhance the detection of enzyme conjugates when used in place of chromogenic substrates (1, 2). As a result, these chemiluminescent enzyme substrates have been incorporated in nonisotopic hybridization assays (1). While nonisotopic techniques have been applied to the detection of HBV DNA in serum (8), nonradioactive RNA probes have not been described for the detection of HDV RNA. In the present study, we report the use of a highly sensitive digoxigenin-labeled RNA probe in combination with the chemiluminescent substrate adamantyl-1,2-dioxetane phenyl phosphate (PPD) for the detection of HDV RNA in serum. Following a severe outbreak of HDV infection, serum samples were collected from the Yucpa Indians in western
Venezuela as part of a prospective epidemiological study of chronic hepatitis B surface antigen (HBsAg) carriers (5). Sera were collected and assayed for HBsAg, anti-HBsAg, hepatitis B soluble e antigen (HBeAg), and anti-HBeAg (Abbott Laboratories, North Chicago, Ill.). Total antibody to HDV (antiHDV) and anti-HDV immunoglobulin M were measured by using kits from Noctech Ltd. (Dublin, Ireland). A total of 200 serum samples were tested by dot blot hybridization using a 32P-labeled RNA probe. From this collection, a panel was assembled which consisted of eight serum samples from HBsAg carriers superinfected with HDV (HBsAg positive and anti-HDV positive) who showed evidence of liver disease and who were positive for HDV RNA and four serum samples from HBsAg carriers who showed no evidence of HDV infection (HBsAg positive and anti-HDV negative; HDV RNA negative). The panel was retested for HDV RNA by the chemiluminescent hybridization assay. HDV RNA probe was prepared by in vitro transcription of linearized pSPT18 (Pharmacia, Uppsala, Sweden) containing a 1-kb EcoRI-PstI restriction fragment. This fragment spans nucleotide positions 775 through 0 to nucleotide 138 and was derived from HN88 (7), which was a gift from M. M. C. Lai (University of Southern California, Los Angeles). Antigenomic-sense transcripts were labeled with either [a-32P]CTP (specific activity, 3,000 Ci/mmol) or digoxigenin-11-UTP by using commercial kits supplied by Boehringer Mannheim Biochemicals (Indianapolis, Ind.). Radiolabeled transcripts were used within 24 h; however, digoxigenin-labeled RNA probes were routinely stored at - 70°C for at least 4 months, and longer storage without loss of activity has been reported
(6). For nucleic acid extraction, 100 ,u of sera was added to an equal volume of lysing solution (100 mM Tris hydrochloride [pH 8.0], 400 mM NaCl, 20 mM EDTA, 4% sodium dodecyl sulfate [SDS], 20 mM vanadyl-ribonucleoside complex, 200 ,ug of proteinase K per ml) and incubated at 65°C for 1 h. After phenol-chloroform extraction and ethanol precipitation, nucleic acids were resuspended in 75 [lI of RNase-free TE (10 mM Tris hydrochloride [pH 8.0], 1 mM EDTA) and denatured by adding 25 RI of formaldehyde (pH 4.5). Samples were incubated at 65°C for 15 min, chilled on ice, and applied to Nytran membranes (Schleicher & Schuell, Keene, N.H.) equilibrated in 20x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) by using the manufacturer's dot blot manifolds. The membrane was UV cross-linked at 0.12 J/cm2 by using a
* Corresponding author. Mailing address: Hepatitis Branch, Centers for Disease Control, 1600 Clifton Rd. A-33, Atlanta, GA 30333. Phone: (404) 639-2354. Fax: (404) 639-1563.
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FIG. 1. Dot blot assay of fivefold dilutions of homologous HDV cDNA hybridized to a digoxigenin-labeled HDV RNA probe (A) and a 37P-labeled HDV RNA probe (B); 1 ng of HBV DNA and I ng of bacteriophage lambda were also used. Exposure times were 60 min for the digoxigenin-labeled HDV RNA probe and 96 h for the 329P-labeled HDV RNA probe.
Stratalinker (Stratagene, La Jolla, Calif.) and then baked at 80°C for 1 h. For hybridization reactions using -32P-labeled RNA probe, membranes were prehybridized and hybridized at 60°C as described by Zinn et al. (10). After hybridization, membranes were washed twice at 68°C for 5 min each time in 2 x SSC-0.1I% SDS and twice at 68°C for 15 min in 0.1 x SSC0.1% SDS. Detection was by autoradiography for 24 to 72 h at - 70°C with intensifying screens. Prehybridization and hybridization reactions using digoxigenin-labeled RNA probe were performed at 60°C as described by Gerritzen and Scholt (4). The probe was used at a concentration of 50 to 80 ng/ml. After hybridization, membranes were washed as described above. Immunologic detection was performed according to the manufacturer's instructions with PPD as the substrate for the anti-digoxigenin-alkaline phosphatase conjugate. Briefly, the membrane was incubated for a minimum of 30 min in a freshly prepared solution of 2% blocking reagent in 100 mM Tris hydrochloride (pH 7.5)-150 mM NaCl. The antibody-enzyme conjugate was diluted to 50 mU/ml in 20 ml of this solution and was incubated with the membrane for 30 min. Unbound antibody conjugate was removed by two washes for 15 min each in 100 mM Tris hydrochloride (pH 7.5)-150 mM NaCl. After a 1-min equilibration in an alkaline buffer (100 mM Tris hydrochloride [pH 9.5], 100 mM NaCl, 50 mM MgCl,), 0.5 ml of substrate-enhancer solution Lumi-Phos 530 (Lumigen, Inc., Detroit, Mich.) was added to the membrane in a heat-sealable bag and the solution was distributed evenly by rolling a pipet over the bag. The membrane was incubated at 37°C for at least 1 h to allow the light emission to reach a steady state and then exposed to Kodak XAR film for 2 to 60 min. To assess the relative sensitivities of the radiolabeled and digoxigenin-labeled probes, each probe was hybridized to replica dot blots containing dilution series of recombinant HDV cDNA (Fig. 1). The chemiluminescent assay for digoxigenin-labeled hybrids consistently detected 0.4 pg with a 60-min exposure (Fig. 1A). To achieve the same level of sensitivity with the radiolabeled probe, an exposure of 96 h was required (Fig. I B). Neither probe hybridized to 1 ng of recombinant HBV DNA or to 1 ng of bacteriophage lambda. The specificity of the digoxigenin-labeled probe was further
3 FIG. 2. Dot blot assay of HDV RNA extracted from sera of HBV carriers and hybridized to digoxigenin-labeled HDV RNA probe (A) and 132P-labeled HDV RNA probe (B). Rows I and 2 contain samples from anti-HDV-positive HBsAg carriers, and rows 3 contain samples from anti-HDV-negative HBsAg carriers. Exposure times were 60 min for the digoxigenin-labeled probe and 7 h for the 32P-labeled HDV RNA probe.
assessed by hybridization with nucleic acids extracted from clinical samples. When extracted sera from eight HDV-superinfected HBsAg carriers were hybridized with the digoxigeninlabeled probe (Fig. 2A, rows 1 and 2), the results that were obtained were identical to those obtained with the radiolabeled probe (Fig. 2B, rows 1 and 2). HBsAg carriers without evidence of HDV infection were found to be negative for HDV RNA by both the radiolabeled probe and the digoxigeninlabeled probe (Fig. 2, row 3 in both panels). Although the sensitivities of the two probes appear to be equivalent, the digoxigenin-labeled probe required only 1 h of development to achieve a signal comparable to that obtained by over 96 h of exposure of the radiolabeled probe. In addition, sera negative for markers of viral hepatitis were negative for HDV RNA by both methods (data not shown). In this study, two hybridization-based assays for the detection of HDV RNA in serum were compared. Anti-genomicsense RNA transcripts labeled with digoxigenin and detected by a chemiluminescent assay and 32P-labeled transcripts detected by autoradiography both showed a detection limit of 0.4 pg. Although additional posthybridization steps are required for the immunodetection of nucleic acids hybridized to the digoxigenin-labeled probe, results were obtained more rapidly because of the short exposure time required when chemiluminescent detection is employed. The RNA probe labeled by either method was specific for HDV sequences and was not found to hybridize to bacteriophage lambda DNA or cloned HBV DNA. False positives were also not observed when the radiolabeled and digoxigenin-labeled probes were hybridized to extracted sera from individuals lacking markers of viral hepatitis or with HBV markers alone. The use of a nonisotopic RNA probe overcomes some of the disadvantages inherent in isotopic detection methods. In vitro transcription reactions typically produce a high yield of RNA transcript; however, when labeled with 32P, the RNA probe must be used within a few days. In contrast, digoxigenin-
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labeled RNA can be stored at 20°C for up to 2 years (6), and the potential hazard of radiation exposure is avoided. In conclusion, the method described here utilizing a digoxigenin-labeled RNA probe and PPD enzyme substrate for the detection of HDV RNA in serum provides a sensitive, safe, stable, and convenient tool for noninvasive detection of HDV -
viremia.
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REFERENCES Beck, S., T. O'Keefe, J. M. Coull, and H. Koster. 1989. Chemiluminescent detection of DNA: application for DNA sequencing and hybridization. Nucleic Acids Res. 17:5114-5122. Bronstein, I., J. C. Voyta, and B. Edwards. 1989. A comparison of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay. Anal. Biochem. 180:95-98. Casacuberta, J. M., R. Jardi, M. Buti, P. Puigdomenech, and B. San Segundo. 1988. Comparison of different non-isotopic methods for hepatitis B virus detection in human serum. Nucleic Acids Res. 16:11834. Gerritzen, A., and B. Scholt. 1990. A nonradioactive riboprobe assay for the detection of hepatitis B virus DNA in human sera. J. Virol. Methods 30:311-318. Hadler, S. C., M. De Monzon, A. Ponzetto, E. Anzola, D. Rivero, A.
6.
7.
8.
9.
10.
Mondolfi, A. Bracho, D. Francis, M. A. Gerber, S. Thung, J. Gerin, J. E. Maynard, H. Popper, and R. H. Purcell. 1984. Delta virus infection and severe hepatitis. Ann. Intern. Med. 100:339-344. Holtke, H.-J., and C. Kessler. 1990. Non-radioactive labeling of RNA transcripts in vitro with the hapten digoxigenin (DIG); hybridization and ELISA-based detection. Nucleic Acids Res. 18:5843-5851. Makino, S., M.-F. Chang, C.-K. Shieh, T. Kamahora, D. M. Vannier, S. Govindarajan, and M. M. C. Lai. 1987. Molecular cloning and sequencing of a human hepatitis delta virus RNA. Nature (London) 39:343-346. Molden, D. P., R. M. Nakamura, H. Suzuki, S. Greer, R. G. Pergolizzi, and C. L. Brakel. 1985. Comparison of radio-labelled DNA-probe with a nonisotopic probe for assay of serum hepatitis B virus DNA. Clin. Physiol. Biochem. 3:174-183. Smedile, A., M. Rizzetto, K. Denniston, F. Bonino, F. Wells, G. Verne, F. Consolo, B. Hoyer, R. H. Purcell, and J. L. Gerin. 1986. Type D hepatitis: the clinical significance of hepatitis D virus RNA in serum as detected by a hybridization-based assay. Hepatology 6:1297-1302. Zinn, K., D. Di Maio, and T. Maniatis. 1983. Identification of two distinct regulatory regions adjacent to the human beta-interferon gene. Cell 34:865-879.
