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Journal of Clinical Microbiology, May 2003, p. 1901-1906, Vol. 41, No. 5
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.5.1901-1906.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

New Enzyme Immunoassay for Detection of Hepatitis B Virus Core Antigen (HBcAg) and Relation between Levels of HBcAg and HBV DNA

Tatsuji Kimura,1* Akinori Rokuhara,2 Akihiro Matsumoto,2 Shintaro Yagi,1 Eiji Tanaka,2 Kendo Kiyosawa,2 and Noboru Maki1

R&D Division, Advanced Life Science Institute, Inc., Wako, Saitama 351-0112,1 Second Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Nagano 390-8621, Japan2

Received 16 August 2002/ Returned for modification 20 January 2003/ Accepted 17 February 2003


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ABSTRACT
 
A new enzyme immunoassay specific for hepatitis B virus (HBV) core antigen (HBcAg) was developed. In order to detect HBcAg, specimens were pretreated with detergents to release HBcAg from the HBV virion and disassemble it to dimers, and simultaneously, the treatment inactivated anti-HBc antibodies. HBcAg detected by the assay peaked with HBV DNA in density gradient fractions of HBV-positive sera. The assay showed a wide detection range from 2 to 100,000 pg/ml. We observed no interference from anti-HBc antibody or blood components, but the assay was inhibited by very high concentrations (>1 µg/ml; corresponding to 80 signal/cutoff) of HBeAg. When the cutoff value was tentatively set at 4 pg/ml, all healthy control (HBsAg and HBV DNA negative, n = 160) and anti-hepatitis C virus-positive (n = 55) sera were identified as negative. HBcAg concentrations correlated very closely with HBV DNA (r = 0.946, n = 145) in 216 samples from 72 hepatitis B patients. In seroconversion panels, HBcAg concentrations changed in parallel with HBV DNA levels. The assay, therefore, offers a simple method for monitoring hepatitis B patients. With a series of sera during lamivudine therapy, HBV DNA levels fell sharply and the HBcAg concentration also decreased, but the change in HBcAg was smaller and more gradual. The supposed mechanism of these changes and their clinical significance are discussed.


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INTRODUCTION
 
Diagnosis of chronic hepatitis B virus (HBV) infection has long been based on HBV serology and measurement of liver enzymes. With the development of therapies for chronic HBV infection including interferon and lamivudine (9, 16), quantitative detection of HBV DNA has been used increasingly as the most important marker for monitoring HBV replication activity and disease progression as well as for assessing responses to antiviral treatment of patients with chronic hepatitis B (7, 8). Several assays for the quantitative measurement of HBV DNA have been developed, such as the branched-chain DNA signal amplification assay (5, 7, 28) and transcription-mediated amplification (TMA)-based (10) or PCR-based (6, 11, 13, 17) nucleic acid amplification assays. However, these methods tend to generate highly divergent results (20, 21, 22, 31) and require cumbersome procedures and expensive equipment, in turn requiring considerable skill and high costs.

On the other hand, immunoassays are generally easy and inexpensive. The nucleocapsid of HBV is composed of either 90 or 120 dimers of HBV core antigen (HBcAg) (3), released into circulation after envelopment. Hence, the quantity of HBcAg in serum would demonstrate virus load as well as HBV DNA. Serum HBcAg assays with specimen pretreatment have been reported previously (4, 29), and the concentration of HBcAg in these assays correlated with levels of HBV-associated DNA polymerase (4). Thus, HBcAg could be a marker for virus load. However, the use of these assays was limited because of relatively low sensitivity and complexity in the procedures.

We have developed an enzyme immunoassay (EIA) for hepatitis B virus core-related antigens (HBcrAg), which reflects HBV load corresponding to HBV DNA (14, 23). The HBcrAg is comprised of HBcAg and hepatitis B e antigen (HBeAg); both are products of precore/core gene and share the first 149 amino acids of HBcAg (25). The HBcrAg assay measures HBcAg and HBeAg simultaneously by using monoclonal antibodies that recognize both denatured HBcAg and HBeAg (14).

In the present study, we developed a new EIA specific for HBcAg. The specimens were pretreated in order to release HBcAg from the virion and to inactivate antibodies before the assay. The correlation between concentrations of HBcAg and HBV DNA was assessed in the sera of hepatitis B patients. With a series of sera from patients undergoing lamivudine therapy, HBcAg concentration decreased less drastically than the HBV DNA level. The supposed mechanism of this difference and its clinical significance are discussed.


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MATERIALS AND METHODS
 
Serum samples and patients. Hepatitis B sera panels were purchased from Boston Biomedica, Inc. (BBI; West Bridgewater, Mass.) or Clinical Science Laboratory, Inc. (CSL; Mansfield, Mass.). Control samples negative for HBV were obtained from blood donors or from chronic hepatitis C patients at the Shinshu University Hospital (Matsumoto, Japan) in 1997. Seventy-two patients with persistent HBV infection (42 males and 30 females [age range, 14 to 82 years]) were examined at least 3 times in 1997, and serum samples were collected 3 times from each patient. Of the 72 patients, 56 showed abnormal levels of serum alanine aminotransferase; the remaining 16 did not and were classified as asymptomatic carriers. None of the 72 patients was treated with antiviral agents such as interferon or lamivudine. Among the 216 sera from the 72 patients, 18 samples indicated an HBcrAg concentration greater than 1 µg/ml, and an additional 3 samples showed HBcAg concentrations of >105 pg/ml. These 21 samples were diluted 10-fold for HBcAg testing.

In addition, two series of serum samples were collected from chronic hepatitis B patients (males, aged 60 and 55 years) who were treated with lamivudine (100 mg/day) between 2000 and 2001.

All sera were stored at -20°C or below until tested. The HBcAg level in the serum samples was stable for at least 7 days at 4°C (data not shown).

Recombinant HBV core-related antigens. Recombinant HBcAg (rHBcAg; amino acids 1 to 183) was expressed in Escherichia coli. The cells were disrupted by sonication and centrifuged. The HBcAg particles in the supernatant were then purified by gel filtration chromatography followed by centrifugation on a sucrose density gradient (30). Recombinant HBeAg (rHBeAg; amino acids -10 to 149) was expressed and purified by the Yeast N-Terminal Expression system (Sigma-Aldrich). The concentrations of these antigens were determined by using the bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, Ill.) and bovine serum albumin standards according to the manufacturer's instructions.

Monoclonal antibodies for HBcAg assay. The establishment of 54 anti-HBcAg monoclonal antibodies and alkaline phosphatase labeling have been previously reported (14).

For the capturing HBcAg, we used HB44, HB61, and HB114 as the immobilized monoclonal antibodies, which recognize denatured HBcAg as well as HBeAg, and were the same as in the HBcrAg assay (14).

For the detection antibody, we selected HB50 monoclonal antibody, which recognizes SPRRR repeats in the C-terminal protamine-like nucleic acid binding domain and is thus specific for HBcAg. All four of the monoclonal antibodies were immunoglobulin G1({kappa}).

Specimen pretreatment and EIA for HBcAg. The HBcAg assay contains a sample pretreatment step in order to release HBcAg from the virion and to inactivate anti-HBc antibodies. A 100-µl specimen aliquot was mixed with 50 µl of pretreatment solution {15% sodium dodecyl sulfate (SDS), 3% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 1% hexadecyltrimethylammonium bromide} and incubated for 30 min at 70°C. This treatment dissociates the HBV capsid from the HBcAg dimers and was confirmed by gel filtration analysis (data not shown).

Microtiter wells (FluoroNunc Black; Nunc, Roskilde, Denmark) were coated with 100 µl of a mixture of anti-HBcAg monoclonal antibodies HB44, HB61, and HB114. Thereafter, the wells were washed, blocked by casein-Na solution, and dried. Pretreated specimen (50 µl) was then added in duplicate to each well filled with 100 µl of assay buffer (pH 8.0). The wells were incubated for 2 h at room temperature and then washed five times. A 100-µl aliquot of alkaline phosphatase-conjugated HB50 monoclonal antibody solution was added to each well and incubated for 1 h at room temperature. After the wells were washed six times, 100 µl of substrate solution (CDP-Star with Emerald II; Applied Biosystems, Bedford, Mass.) was added. The relative luminescence intensity (RLI) was measured with a microplate reader (LUMINOUS CT-9000D; DIA-IATRON, Tokyo, Japan). Chemiluminescence detection gave the assay a broad dynamic range.

In each assay, rHBcAg that had been serially diluted with normal human serum was used as a standard. The standard log RLI was plotted versus the log concentration (Fig. 1), and HBcAg concentrations in each specimen were calculated from the calibration curve.



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  		FIG. 1. Standard curve and detection limit of the HBcAg assay. The rHBcAg standard was diluted in normal human serum and detected by the HBcAg assay. The assay reactivity is shown as the RLI. Broken lines indicate lower and upper detection limits. In the inset, results are means of 6 assays and the error bars show 2 SD. The dotted line indicates the mean + 2 SD of the zero calibrator. Std., standard.

HBcrAg assay. The HBcrAg assay was described previously (14) and was performed similarly to the HBcAg assay. The assay uses HB91 and HB110 monoclonal antibodies as detector antibodies that react with denatured HBcAg and HBeAg; thus, the assay detects precore and core proteins.

HBV markers and HBV DNA measurement. HBeAg, anti-HBe, and anti-HBc were measured by clinically applied radioimmunoassay (Dinabbott, Tokyo, Japan). HBsAg was measured by chemiluminescent immunoassay (Dinabbott). Samples showing values over the detection range were remeasured after dilution to obtain quantitative results.

HBV DNA was detected by TMA (Chugai Diagnostics Science Co., Ltd., Tokyo, Japan), which has a detection range between 3.7 and 8.7 log genome equivalents (LGE)/ml (corresponding to 5 x 103 to 5 x 108 copies/ml), or by PCR (Amplicor HBV Monitor test; Roche Molecular Systems, Inc., Branchburg, N.J.) with a detection range between 4 x 102 and 4 x 107 copies/ml.

In the BBI PHM 935A/B panels, the results for HBV DNA by the Amplicor HBV Monitor test were obtained from the supplier's data sheet.

Sucrose density gradient ultracentrifugation. HBeAg-positive serum (0.1 to 1.0 ml) was layered on a linear 10 to 60% (wt/wt) sucrose gradient, and centrifugation was carried out at 200,000 x g (33,400 rpm) for 15 h at 4°C with a Beckman Sw40Ti rotor. The centrifuged solution was fractionated (40 fractions of 300 µl) by micropipette. The density of each fraction was calculated from the weight and volume. Each fraction was diluted 10-fold and tested for HBcAg as well as for HBsAg, HBeAg, and HBV DNA by PCR.


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RESULTS
 
Detection range, linearity, and reproducibility of the HBcAg assay. We constructed a new EIA specific for HBcAg released from the HBV virion by pretreatment with SDS. In the HBcAg assay, the calibration curve was determined by using the rHBcAg standard serially diluted in normal human serum (Fig. 1). The upper detection limit of this assay was approximately 1 x 105 pg/ml (Fig. 1). The analytical lower detection limit was 2 pg/ml (Fig. 1), as the concentration at which the mean - 2 standard deviations (SD) of the RLI did not overlap with the mean + 2 SD of the zero calibrator (n = 6) (Fig. 1, inset). This assay displayed a broad dynamic range, from 2 to 105 pg/ml.

The linearity of the assay was examined in sera serially diluted in normal human serum (Fig. 2). Two HBeAg-positive sera (CSL P0339/2-02 and -09) were used; the former was anti-HBc positive, and the latter was anti-HBc negative. The quantities of HBcAg decreased linearly with serial dilution on a straight line through the zero point.



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  		FIG. 2. Dilution linearity of hepatitis B sera. Two HBeAg-positive sera were serially diluted in normal human serum and measured by the HBcAg assay. •, P0339/2-02; {square}, P0339/2-09.

Intra-assay reproducibility was assessed from 10 measurements of three specimens. The mean HBcAg values of the specimens were 3.0 x 102, 4.1 x 102, and 1.3 x 104 pg/ml, and the coefficients of variation were 9.3, 16.9, and 5.4%, respectively.

Distribution of HBcAg in sucrose density fractions. Seven sera positive for HBV DNA were subjected to ultracentrifugation on a 10 to 60% (wt/wt) sucrose density gradient, and fractions were diluted and tested for HBcAg, HBsAg, HBeAg, and HBV DNA by PCR. The results for a serum sample are shown in Fig. 3. HBcAg appeared in fractions around a density of 1.12 g/ml and peaked at fraction 25, as did HBV DNA. HBsAg was distributed in fractions of lower density (1.09 g/ml) while HBeAg was dispersed widely in fractions of even lower density. The same results were observed in the other six sera. These data provide support for the hypothesis that the assay indeed detects nucleocapsid protein specifically.



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  		FIG. 3. HBcAg distribution in density gradient fractions. An HBV DNA-positive serum was subjected to ultracentrifugation on a 10 to 60% (wt/wt) sucrose density gradient. Fractions were diluted 10-fold and tested for HBcAg ({circ}) as well as for HBsAg ({blacktriangleup}), HBeAg ({square}), and HBV DNA ({blacksquare}) by PCR. Data are expressed as percentages of peaked value. The density of each fraction is shown as a broken line.

Recovery of the HBcAg assay. Aliquots of 10 µl of HBcAg-positive serum (BBI PHJ201-03 and -07) were added to 90 µl of reference serum. A normal human serum and four hepatitis B sera (CSL P0339/2-01, -03, -04, and -08) were utilized as reference sera. Three of these hepatitis B sera (CSL P0339/2-01, -03, and -04) were anti-HBc positive. No HBcAg was detected in any reference sera. The antigen measured in these samples was designated as recovered HBcAg, and recovery rates were calculated as ([recovered HBcAg]/[added HBcAg]) x 100% (Table 1). Recovery rates ranged from 79 to 121%, and there were no significant differences between anti-HBc antibody positivity and negativity among the samples. These data indicate that anti-HBc did not interfere with the HBcAg assay.


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  		TABLE 1. Recovery of HBcAga

Interference of the HBcAg assay. The influence of various blood elements was assessed by using an interference check kit (International Reagents Corporation, Kobe, Japan). Five concentrations of each blood element were added to two samples of HBcAg-positive sera, and HBcAg was measured. Unconjugated bilirubin (<19 mg/dl), conjugated bilirubin (<20 mg/dl), hemoglobin (<440 mg/dl), chyle (<2,350 formazin turbidity units), and immunoglobulin M rheumatoid factor (<50 IU/ml) did not interfere with this assay (data not shown).

Because the HBcAg assay uses monoclonal antibodies that react to both HBcAg and HBeAg as the immobilized antibody, HBeAg may interfere with the HBcAg assay. The influence of HBeAg was assessed by using rHBeAg. rHBcAg (0.1, 1, 10, or 100 ng/ml) was mixed with rHBeAg (1, 10, or 100 ng/ml or 1 or 10 µg/ml) and measured by the HBcAg assay. Regardless of the rHBcAg concentration, no significant influence (-10 to +9%) was observed when 1 µg or less of rHBeAg/ml was added. However, considerable (32 to 43%) inhibition was observed when 10 µg of rHBeAg/ml was added (data not shown). So, before performing the HBcAg assay, we measured HBcrAg, which contains HBcAg and HBeAg, and if the samples showed over 1 µg of HBcrAg/ml, we assessed for HBcAg after a 10-fold dilution in normal human serum. rHBeAg at 10 and 100 ng/ml and at 1 and 10 µg/ml corresponds to 1.4, 12.4, 79.6, and 166.0 signal/cutoff (s/co) by clinically applied radioimmunoassay, respectively.

HBcAg in normal and hepatitis C serum samples. HBcAg was examined by using healthy control (HBsAg and HBV DNA negative, n = 160) sera and sera of hepatitis C patients (anti-hepatitis C virus [HCV] positive, n = 55). HBcAg levels were below 3.9 pg/ml (mean = 0.76 pg/ml; SD = 0.65 pg/ml) and 2.4 pg/ml (mean = 0.37 pg/ml; SD = 0.50 pg/ml), respectively. Based on these data, we set a tentative cutoff for HBcAg positivity at 4 pg/ml. These data indicate high specificity of this assay for HBcAg.

Correlation with HBV DNA. HBcAg was examined in 216 sera from 72 hepatitis B patients. HBV DNA was also measured by TMA. When the cutoff was set at 4 pg/ml, 155 of 216 samples were HBcAg positive, whereas TMA identified 178 samples as positive for HBV DNA. The correlation between the log concentration of HBcAg and that of HBV DNA is shown in Fig. 4. The correlation coefficient was 0.946 for sera with HBcAg positive and HBV DNA quantitative results (n = 145), indicating the results of the HBcAg assays correlated very well with HBV DNA.



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  		FIG. 4. Correlation between the concentrations of HBcAg and HBV DNA in sera of hepatitis B patients. HBcAg was examined in 216 serum samples from 72 hepatitis B patients. HBV DNA was also measured by TMA (detection limit, 3.7 to 8.7 LGE/ml). •, HBeAg positive; {circ}, HBeAg negative. Broken lines indicate the lower or upper limits of the assays.

HBcAg in seroconversion panels. HBcAg was examined in commercially available BBI PHM935A/B seroconversion panel sera that were sets of serial bleeds taken from a donor (Fig. 5A). The HBcAg was detectable during days 21 to 121, and the HBcAg level peaked at day 68. The HBcAg concentration changed in parallel to the HBV DNA level (Amplicore HBV Monitor test).



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  		FIG. 5. Changes in HBcAg and HBV DNA concentrations in series sera. (A) BBI PHM935A/B seroconversion panels. {circ}, HBcAg [log (picograms/milliliter)]; {blacksquare}, HBV DNA detected by the Amplicore HBV Monitor test (detection limit, 4 x 102 to 4 x 107 copies/ml). The HBV DNA concentrations on days 68 and 85 were over the detection range (>4 x 107 copies/ml). (B and C) Sera of a chronic hepatitis B patient who was treated with lamivudine. {circ}, HBcAg [log (picograms/milliliter)]; {blacksquare}, HBV DNA detected by TMA (detection limit, 3.7 to 8.7 LGE/ml). The gray bar indicates the period of lamivudine administration.

HBcAg and HBV DNA in series sera of an HBV patient during lamivudine therapy. HBcAg and HBV DNA TMA were examined in series sera of chronic hepatitis B patients who were treated by lamivudine (Fig. 5B and C). Changes in the HBcAg concentration nearly paralleled those of HBV DNA before lamivudine treatment had begun. After lamivudine administration had started, HBV DNA levels fell sharply while HBcAg concentrations also decreased, but the change in HBcAg was smaller and more gradual.


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DISCUSSION
 
Recently, HBV DNA TMA and PCR have been widely used to monitor virus load (6, 8, 10, 13, 17, 20, 21). However, conventional EIA has some advantages over nucleic acid amplification assays. EIA is a relatively simple method and provides a low cost and quantitative analysis with high reproducibility.

Measurement of the HCV core antigen after specimen pretreatment has been reported as useful for diagnosing and monitoring hepatitis C (12, 15, 18, 26). A highly sensitive EIA was previously developed for the HCV core antigen (1, 2, 27) in which the HCV core antigen was released from the virion by SDS pretreatment prior to EIA detection. We have applied this detection system to HBV. In a previous study, an EIA for HBcrAg was developed (14, 23) that measures HBcAg and HBeAg simultaneously by using monoclonal antibodies that recognize denatured HBcAg and HBeAg commonly. The level of HBcrAg reflects the HBV load nearly as well as HBV DNA (14, 23).

In the present study, we developed an EIA specific for HBcAg, which includes a single-step pretreatment with SDS. In density gradient fractions, HBcAg peaked with HBV DNA (Fig. 3), showing the assay detected the HBcAg of the HBV virion in serum. As a result of pretreatment, the HBcAg was released from the virion and dissociated to HBcAg dimers that were confirmed by gel filtration analysis (data not shown). The pretreatment also inactivated anti-HBc antibody in specimens, and therefore, HBcAg could be measured quantitatively even in anti-HBc-positive specimens. This was confirmed by the recovery test with anti-HBc-positive sera (Table 1).

HBeAg shares an identical 149-amino-acid sequence with HBcAg (25). The immobilized monoclonal antibodies of the HBcAg assay capture not only HBcAg but also HBeAg. Thus, theoretically, HBeAg would inhibit the HBcAg assay. However, the inhibition was observed only at very high concentrations (>1 µg/ml, corresponding to 80 s/co) of HBeAg. In order to avoid the inhibition and scale out, a sample dilution was needed for 21 of 217 serum samples from hepatitis B patients. Moreover, the inhibition rate was 32 to 43% at 10 µg of HBeAg/ml (corresponding to 166 s/co). This is lower by only 0.16 to 0.24 log units. The results of the HBcAg assay were not seriously affected by HBeAg.

HBcAg concentrations and HBV DNA levels were very closely correlated in hepatitis B patients (r = 0.946) (Fig. 4). The correlation between HBcAg and HBV DNA levels was better than that of HBcrAg versus HBV DNA (14, 23), as might be expected for a capsid protein-specific assay. HBcAg levels also paralleled HBV DNA levels in seroconversion panel sera (Fig. 5A) and in serum from a hepatitis B patient before lamivudine therapy (Fig. 5B and C). Thus, the HBcAg assay could be a virus load marker alternative to HBV DNA assays.

The lower and upper detection limits of the HBcAg assay were 2 and 105 pg/ml, respectively (Fig. 1), which corresponded to approximately 5 and 9 LGE of HBV DNA/ml (105 and 109 copies/ml) (Fig. 4). This broad dynamic range of over 4 orders of magnitude is appropriate for quantitative detection of HBV virus loads that vary over a wide range. The HBcAg assay was as sensitive as HBV DNA branched-chain DNA but less sensitive than HBV DNA TMA or PCR. However, particularly in areas in which TMA or PCR is not widely used, the virus load marker measured by simple EIA would be needed for blood screening of HBV infection as well as for virus load monitoring. The HBcAg assay also could be used to screen for HBsAg-negative HBV infection.

In the series sera, changes in HBcAg concentrations nearly paralleled those of HBV DNA prior to medical treatment. To the contrary, a decrease of HBcAg concentrations was much slower than that of HBV DNA concentrations during lamivudine administration (Fig. 5B and C). Lamivudine, a nucleoside analogue, strongly inhibits HBV reverse transcriptase and thus lowers HBV DNA production rapidly (19, 24). However, lamivudine has little or no effect on covalently closed circular DNA (16) and does not inhibit virus DNA transcription to mRNA or its translation. We hypothesized that during lamivudine treatment, a considerable amount of HBV covalently closed circular DNA remains in the liver, and HBcAg would therefore be translated and released into circulation as empty virus (HBV DNA defective virus-like particle). We verified this to some extent (unpublished data).

If this hypothesis is true, HBcAg levels could reflect HBV levels remaining in the liver. Hence, the measurement of HBcAg in addition to that of HBV DNA may be useful for estimating the efficacy of lamivudine treatment or the risk of the emergence of YMDD variants and for deciding whether to discontinue administration of lamivudine. To confirm these preliminary observations, additional clinical and diagnostic studies of much larger populations are required.


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ACKNOWLEDGMENTS
 
We are grateful to Yoko Oda and Sanae Nakatsuka for the establishment of monoclonal antibodies and technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: R&D Division, Advanced Life Science Institute, Inc., Wako, Saitama 351-0112, Japan. Phone: 81 48 465 2765. Fax: 81 48 465 2761. E-mail: tkimura{at}alsi-i.co.jp. Back


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Journal of Clinical Microbiology, May 2003, p. 1901-1906, Vol. 41, No. 5
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.5.1901-1906.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kimura, T., Ohno, N., Terada, N., Rokuhara, A., Matsumoto, A., Yagi, S., Tanaka, E., Kiyosawa, K., Ohno, S., Maki, N. (2005). Hepatitis B Virus DNA-negative Dane Particles Lack Core Protein but Contain a 22-kDa Precore Protein without C-terminal Arginine-rich Domain. J. Biol. Chem. 280: 21713-21719 [Abstract] [Full Text]  

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