Journal of Virologicai Methoa!s, 34 (1991) 233-243 0 1991 Elsevier Science Publishers B.V. / All rights reserved / 01660934/91/%03.50 ADONZS0166093491004718


VIRMET 01225

An immunoassay

for specific amplified HCV sequences

Luisa Imberti’, Elisabetta Cariani’, Alessandra Bettinardi’z Antonella Zonaro’, Albert0 Albertini* and Daniele Primi’ ‘Consorzio per le Biotecnologie, Consiglio Nazionale delle'Ricerche (CNR) and ‘Institute of Chemistry, School of Medicine. University of Brescia. Brescia, Italy (Accepted

13 May 1991)

The direct detection of viraemia could improve greatly the efficacy of presently available assays. Due to its sensitivity, the polymerase chain reaction represents the method of choice for direct detection of viral nucleic acid, However, the clinical application of this method is hampered by the requirement of hybridization with radioactively labelled probes. In this study we demonstrate that HCV cDNA, amplified by the polymerase chain reaction from both liver tissues and sera, can be detected specifically by a new nonradioisotopic method, DNA enzyme immunoassay, that is based on an antibody that selectively recognizes double, but not single-stranded DNA. The assay reveals the hybridization events, independently from the DNA sequences, and therefore can be used with any combination of primers and probes. Most importantly, the method has a conventional ELISA format and is compatible with standard facilities of clinical laboratories. The availability of this new approach for revealing amplified sequences may facilitate greatly the use of PCR as the method of choice for early diagnosis of HCV infection. DNA

enzyme immunoassay; HCV infection; Polymerase chain reaction

*Note: On leave of absence from the Unitk d’Immunochimie Analytique, Institut Pasteur, 28 rue Dr. Roux, 75124 Paris, France. Correspondence to: D. Primi, Consorzio per le Biotecnologie, Laboratorio di Biotecnologie, P. le Spedali Civili, 1, 25123 Brescia, Italy.


Introduction Hepatitis C virus is the etiological agent of most non-A, non-B (NANB) hepatitis (Kuo et al., 1989; Van der Poe1 et al., 1989, 1990), a disease that probably accounts for more than 50% of chronic hepatitis and which progresses to cirrhosis in about 20-30% of the cases (Dienstag, 1983; Koretz et al., 1985; Alter, 1988, 1989; Choo et al., 1989). The cloning and sequencing of the HCV genome has made it possible to develop an assay for anti-HCV antibodies (Kuo et al., 1989; Miyamura et al., 1990). This immunoassay has markedly diminished the incidence of posttransfusional hepatitis but, because of the long delay required for seroconversion (Alter et al., 1989; Alter and Sampliner, 1989), this test is not sufficient to remove completely the risk of this disease (Alter et al., 1989). The most sensitive tests for the diagnosis of HCV are those based on molecular hybridization, permitting the direct detection of HCV RNA. A more timely diagnosis of HCV infection can be obtained by the polymerase chain reaction (PCR), which allows the detection of viral nucleic acid in liver and plasma in very low con~ntration (Garson et al., 1990; Kato et al., 1990; Weiner et al., 1990). The specificity of HCV RNA amplified by PCR is generally verified by Southern blot hybridization. Since this method needs the labelling of probe or primers with radioisotopes, the detection of HCV RNA in NANB hepatitis is still confined to research laboratories. We describe a ,new method that combines the sensitivity of PCR with the simplicity and versatility of conventional immunoassay. This may permit the development of routine diagnostic assays for the identification of HCV RNA in liver and serum. This immunoassay will allow not only the screening of large number of samples, but may be also used to identify acute infection and to detect viraemia and persistent infection.

Patients and Methods Patients and specimens Liver tissues were obtained by percutaneous biopsy taken for histological diagnosis from 9 Italian adult patients, 8 of whom were anti-HCV antibody positive (Ortho EIA and Ortho RIBA, Ortho Diagnostics, Raritan, NJ) and one anti-HCV antibody negative. Among the anti-HCV antibody positive patients, one had chronic persistent and 7 chronic active hepatitis. All these patients were negative for hepatitis B surface and ‘e’ antigens and none had developed antibodies to the core antigen (HBcAg). The immunological and clinical profiles of these subjects are shown in Table 1. Liver specimens and serum samples, collected on the same day of the biopsy, were stored at - 80°C before PCR analysis.


Synthesis of specific oligonucleotides

To perform nested PCR two sets of oligonucleotide primers were used, both located in the 5’ untranslated region (UTR) of HCV (Okamoto at al., 1990). The sequences of the primers were: (a) outer primers 1A (S-GATGCACGGTCTACGAGACCTC-3’, antisense; position from - 1 to - 21) and 1B (S-AACTACTGTCTTCACGCAGAA-3’, sense; position from - 289 to - 269) and (b) inner primers 2A (S-GCGACCCAACACTACTCGGCT-3’, antisense; position from - 70 to -90) and 2B (5’-ATGGCGTTAGTATGAGTG-3’, sense; position from -257 to -240). Oligonucleotides 3A (5’AGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCT-3’; position from - 185 to - 143) and 3B (5’-AGGTGAGTACACCGGAATTGC-3’; position from - 185 to - 166) derived from a region internal to the PCR product and not overlapping the primers used for the amplification, were also synthesized and used as probes. All primers were prepared using a 391 DNA Synthesizer, Applied Biosystem, Santa Clara, CA. Preparation of RNA and cDNA synthesis

Total RNA was prepared from 200 ~1 of sera and from liver specimens by guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski and Sacchi, 1987). HCV RNA was transcribed to cDNA by reverse transcriptase, according to the manufacturer’s instruction (Riboclone cDNA Synthesis System, Promega Corp., Madison, WI) using the antisense primer 1A.

TABLE 1 Clinical and histological Patient


features of patients with chronic hepatitis


Liver histology

ALT WJ/ml)




25 45 61 50 19 60 55

8’ 9

24 60

: 4 5’

M L F L F :

*History of drug addiction. CIRR = cirrhosis.

150 CAH 306 CAH CPH 1:; CAH. 145 CAH 158 CAH 30 post-hepatitic residual alterations 315 CAH 185 CAH and CIRR

antibody RIBA

Duration of clinical history (years)

+ + + + + + + +

CAH = chronic active hepatitis; CPH = chronic persistent




of cDNA by PCR

Nested PCR (Garson et al., 1990) was carried out in two steps on 5 ~1 of the cDNA obtained from serum and liver. Both steps were carried out in a final volume of 100 ~1 containing 67 mM Tris-HCl, pH 8.8, 16.6 mM (NH&S04, 1.5 mM MgC12, 10 mM 2-mercaptoethanol, 100 pg/ml bovine serum albumin, 4 units of DNA polymerase from thermus thermophilus HB8 (Tth DNA polymerase; Toyobo Co., Osaka, Japan), 200 mM of each dNTP, 50 pmol of each primer and 5 ~1 of cDNA. The mixture was subjected to PCR amplification using the Perkin-Elmer Thermal Cycler (Norwalk, CT). The first step consisted of 35 cycles (denaturation, 94°C for 60 s; annealing of primers, 45°C for 60 s; extension, 72”C, for 120 s) and was performed with primers 1A and 1B. In the second step, 3 ~1 of the first PCR products were amplified, using the same protocol, with primers 2A and 2B, for another 25 cycles. After removing the layer of mineral oil, an aliquot of all amplification reactions was analyzed by electrophoresis in 1.5% agarose gels. Bands were visualized by ethidium bromide staining and photographed at 302 nm. Southern blots Amplified HCV DNA was run on agarose gels and transferred to Hybond N+ nylon blotting membrane (Amersham, Amersham, U.K.), following the manufacturer’s instruction. Filters were incubated for 1 h at 42°C in prehybridization solution containing 5 x SSC (1 x SSC = 0.15 M NaCl, 0.013 M Na Citrate), 5 x Denhardt’s solution (1 x Denhardt’s = 0.02 polyvinylpyrrolidone, 0.02% Ficoll, 0.02% BSA), 0.5% SDS, 100 pg/ml of denatured salmon sperm DNA, and then hybridized overnight at 42°C with 32P-labelled (abo ut 1 x lo6 cpm/ml) oligonucleotide probe 3B. The membrane was washed twice for 15 min at room temperature in 2 x SSC, 2% SDS and then for 30 min at 42°C in 0.2% SSC, 0.2% SDS and autoradiographed overnight at -70°C. DNA molecular weight markers (Boehringer Biochemia Robin, Milan, Italy) were applied to each gel in order to determine the size of the resulting bands. Production

of 27-14-09


Spleen cells from a lo-months-old non immunized autoimmune MLR/lpr mouse were fused with SP2/0 myeloma cell line. Supernates were tested for the presence of antibodies specific for native or denaturate DNA. Only those hybridomas displaying activity against native DNA, but not against denaturate DNA were selected. One of these hybridomas, 27-14-D9, appeared to best discriminate between duplex DNA and single stranded target and was therefore used for the present study. The immunoglobulin fraction used in these experiments was pre ared as follows: 300 ml of supernates from a culture containing 2 x 10g cells of 27-14-D9 hybridoma were harvested and


concentrated 10 times. The protein fraction containing specific antibodies was precipitated with sodium sulphate, 180 g/l. After centrifugation, we resuspended the pellet in 15 ml of sodium chloride, 9 g/l and extensively dialyzed it against phosphate-buffered saline. The immunoglobulin-enriched solution was then aliquoted and stored at -20°C until used. For biochemical characterization, we adsorbed 4 ml of the concentrated supernate on protein ASepharose (Pharmacia, Uppsala, Sweden) and eluted the specific antibodies with glycineC1 buffer, 100 mmol/l, pH 4. DNA enzyme immunoassay (DEZA)

Quantification of the PCR products was carried out using the DEIA assay as described previously (Mantero et al., 1991). Briefly, streptavidin coated microtitre plates, kindly provided by Sorin Biomedica (Saluggia, Italy), were incubated overnight at 4°C with 10 ng/well of biotinylated 3A oligonucleotide in 100 ~1 of TE (Tris 10 mM, pH 8.0, EDTA 1 mM, pH 8.0). The solid phase was washed 5 times with 200 ~1 of washing solution containing 6.7 mM phosphate buffer, pH 6.4, 0.13 M sodium chloride, 0.004% Cialit (Sigma Chemical Co., St. Louis, MO) 0.1% Tween 20. The crude PCR mixtures were denatured on a heat block for 10 min at 100°C and then quickly cooled on ice. Twenty-five ~1 of the PCR product, diluted 4 times in hybridization solution (1 x SSC, 2 x Denhardt’s solution, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA), were added to the coated wells and incubated for 1 h at 50°C. After 5 washes with washing solution, each well received 100 ~1 of a l/ 100 dilution in PBS- 10% fetal calf serum of a standard preparation of the 27-14-D9 mAb, specific for double-stranded DNA. After 2 h incubation at 37°C and 5 washes with washing solution, the bound antibody was revealed with 100 ~1 of HRPlabelled rabbit anti-mouse IgG antibody (ICN Biochemical, High Wycombe, Bucks, U.K.) diluted l/20000 in PBS 10% fetal calf serum. Following 1 h incubation at room temperature and 5 washes, the wells received 100 ~1 of the chromogen/substrate solution (0.1 M citrate buffer, pH 5, containing ophenylenediamine hydrochloride and 10 ~1 of HzOz) and the calorimetric reaction was allowed to develop for 30 min at room temperature in the dark. After blocking with 200 ~1 of 1 N sulphuric acid the net absorbance was read in a microtitre plate reader at 450 nm. Results Detection and quantification of HCV in biological samples

The aim of the study was to develop a sensitive and rapid test for the detection of HCV sequences in liver and serum of patients with chronic hepatitis. To this end, RNA isolated from hepatic tissue of 9 patients with chronic hepatitis, 8 of whom were positive for HCV antibodies, was used to


synthesize complementary DNA. This material was then used as template for nested PCR reactions. The 2 sets of primers (lA, lB, 2A and 2B) used for this analysis encompass part of the 5’ UTR of HCV, which is highly conserved among different HCV isolates (Okamoto et al., 1990). With 7 of the 9 samples analyzed we obtained, both in the first and second steps of amplification, PCR products of the expected size (290 and 187 base pairs, respectively). The specificity of the amplified cDNAs obtained after the second step of amplification was firstly analyzed by Southern blot hybridization with a 32Plabelled internal probe (Fig. 1, bottom). The signals generated by seven samples had the same electrophoretic mobility of the amplified products, confirming therefore the presence of HCV sequences in these biopsies. Aliquots of the amplified samples were also analyzed by a calorimetric assay (DEIA), recently developed in our laboratory, able to reveal specifically PCR products. The results, obtained by DEIA, using the immobilized sense 3A primer (Fig. 1, top), demonstrate that there is a perfect correspondence between the optical absorbance values and the intensity of the bands obtained in Southern blot.

167 bp + +






Fig. 1. Comparative analysis of amplified HCV cDNA from liver of 9 patients with chronic hepatitis. Aliquots of the same PCR products were specifically revealed by Southern blot hybridization (bottom) and by DEIA (top). The DEIA values are expressed as optical density values at 405 nm. The cut-off value was 0.20 and represents the mean value of 10 negative controls + 3 SD. The positive control (+) used in these experiments was a serum from an HCV-infected patient that contains high titre of anti-HCV antibodies. Samples with asterisk were diluted 1 to 2 before analysis by DEIA.










PATIENTS # Fig. 2. Comparative analysis of amplified HCV cDNA from sera of 9 patients with chronic hepatitis. Aliquots of the same PCR products were specitkally revealed by Southern blot hybridization (bottom) and by DEIA (top). The DEIA values are expressed as optical density values at 405 nm. The cut-off value was 0.20 and represents the mean value of 10 negative controls + 3 SD. The negative control (-) used in these experiments was a serum from a normal healthy donor.

Interestingly samples #5, #6 and #S gave an optical density value in the plateau range and needed to be diluted 1:2 before the assay. Thus, the differences in the intensity of the signals in the different samples are likely to reflect true variations in the amounts of viral sequences present rather than artifacts connected with the DNA transfer procedure in Southern blot. The specificity of the assay was further confirmed by the optical density values obtained with samples #2 and #7 that were clearly below the cut-off value. To verify the possibility of a more wide applicability of the method for the screening of blood donors, we employed the combination of PCR and DEIA test for the detection of HCV in serum samples obtained from the same patients analyzed for the presence of HCV sequences in the liver. As shown in Fig. 2, HCV nucleic acids were detected in the sera of the same patients that had positive biopsies. Even in this case there was a perfect correspondence between the optical density values obtained by DEIA and the intensity of the Southern hybridization bands. Taken collectively the results establish that DEIA may represent an ideal alternative to conventional hybridization methods to reveal specifically amplified DNA sequences.


Discussion Recently, PCR has allowed the detection of HCV RNA in chimpanzee sera during the acute phase of the illness and in human liver and serum samples (Shimuzu et al., 1990; Weiner et al., 1990). This technique represents an important advance in the field of diagnostic research because even minute amounts of nucleic acid present in a sample can be ampli~ed (Saiki et al., 1988). One limitation of PCR is that it cannot be used easily in the clinical laboratory, principally because the determination of the specificity of the amplified products requires the use of radioisotopes. Several isotopic and nonisotopic methods for detecting hybrids between oligonucleotide probes and amplified DNA have been described (Syvanen et al., 1988; Cheab and Kan, 1989; Keller et al., 1989; Kemp et al., 1989; Saiki et al., 1989; Ballabio et al., 1990; Coutlee et al., 1990). Most of these methods involve labelling the probe or the PCR primers with molecules acting as specific ligands for enzyme-labelled antibodies or other macromolecules. However, these systems require the preparation of labelled reagents specific for each target DNA and follow analytical schemes that often are not fully compatible with those used in routine diagnostic laboratories. To overcome this problem we developed an immunoassay (DEIA) that allows the rapid and specific detection of amplified target sequences. The test is based on a monoclonal antibody that recognizes double stranded, but not single stranded DNA. This molecule, therefore, reveals the event of hybridization generated by the interaction between an immobilized capture probe and the specific amplified product. The 27-14-D9 mAb does not react with a specific probe immobilized on microwells through an avidin-biotin bridge, nor with non-specific amplified sequences, since they are removed by washes. We have previously shown that this test, when combined with PCR, is extremely sensitive and allows the identification in serum samples of as few as 2 hepatitis B viral genomes (Mantero et al., 1991). Since the assay reveals the hybridization event, independently from the DNA sequences and does not require primers or DNA target modi~cations, it can theoretically be used for the analysis of any amplified product. In this study we compared the efficiency of conventional Southern blot hybridization with a 32P labelled probe with DEIA and demonstrated that there is a complete correspondence between the results obtained with the two methods. In patients #2 and #7 both methods failed to reveal the presence of HCV sequences in liver tissue and in serum, but we could demonstrate the presence of HCV sequences in liver and in sera of all the other patients. One of the 2 negative samples belonged to an anti-HCV antibody positive patient (#2), while the other one derived from an anti-HCV antibody negative subject (patient #7). The high degree of conservation of the region used as a PCR template strongly suggests that the failure to reveal HCV nucleic acids in the former sample is not due to the presence of an HCV variant. Thus, the relationship between the PCR results and the clinical manifestations accompanied by seroconversion in this patient remains unclear.


The results indicate that the analysis of serum samples is as sensitive as the direct detection of HCV RNA in liver tissue. Therefore, the use of DEIA test should facilitate greatly the direct assay of serum HCV RNA in patients with chronic hepatitis, despite the low amounts of circulating viral particles. The possibility of detecting target nucleic acid in sera with DEIA opens new interesting possibilities for large scale screening of blood donors. Preliminary results suggest that this technology may also be valuable for defining the time course of the viraemia in infected subjects and may allow rapid diagnosis of acute hepatitis C, weeks or months before diagnosis by serological methods is possible. In this study, HCV sequences were analyzed after nested PCR in order to exclude the possibility of false negative results. However, we ,obtained evidence that the DEIA assay can easily reveal amplified HCV speciftc cDNA after 35 cycles of amplification with only one set of primers. One of the major advantages of the DEIA test is that it provides optical density values and is therefore an ideal analytical assay for use of future development of quantitative measurement of PCR products for direct assessment of viral replication. The successful application of the DEIA assay for the detection of HCV sequences has indeed proved its versatility and the advantage that it can offer over more classical hybridization methods. Furthermore, the method can be automated with conventional instrumentation already available in clinical laboratories. Thus, the association between PCR and DEIA may have wide application for the diagnosis and prevention of hepatitis C. This technology can be used for blood donor screening and for epidemiological studies using linked donors and recipient samples obtained in prospective studies of transfusion associated hepatitis. The availability of this new method for early identification of infected individuals should also permit the introduction of early antiviral treatment and monitoring of patients with chronic non-A, non-B hepatitis infection. Acknowledgements

This work is supported by Sorin Biomedica, Saluggia, Italy. E.C., A.B. and A.Z. are supported by fellowships of Fondazione Golgi, Brescia, Italy. References Alter, H.J. (1988) Transfusion-associated non-A, non-B hepatitis: the first decade. In: A.J. Zuckerman (Ed.), Viral Hepatitis and Liver Disease, pp. 357-342. Alan R. Liss, New York. Alter, H.J. (1989) Chronic consequences of non-A, non-B hepatitis. In: L.B. Seeff and J.H. Lewis (Eds.), Current Perspectives in Hepatology, pp. 83-97. Plenum Medical, New York. Alter, H.J., Purcell, R.H., Shih, J.W., Melpolder, J.C., Houghton, M., Choo, Q.-L. and Kuo, G. (1989) Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N. Engl. J. Med. 321, 14941500.

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An immunoassay for specific amplified HCV sequences.

The direct detection of viraemia could improve greatly the efficacy of presently available assays. Due to its sensitivity, the polymerase chain reacti...
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