Sensitive Enzyme Immunoassay for Hepatitis B Virus Core-Related Antigens and Their Correlation to Virus Load

Abstract

A sensitive enzyme immunoassay (EIA) specific for hepatitis B virus core antigen (HBcAg) and hepatitis B e antigen (HBeAg) was developed. We designated the precore/core gene products as hepatitis B virus (HBV) core-related antigens (HBcrAg). In order to detect HBcrAg even in anti-HBc/e antibody-positive specimens, the specimens were pretreated in detergents. The antibodies are inactivated by this pretreatment and, simultaneously, the antigens are released and the epitopes are exposed. The assay demonstrated 71 to 112% recovery using HBcrAg-positive sera. We observed no interference from the tested anticoagulants or blood components. When the cutoff value was tentatively set at 10 3 U/ml, all healthy control (HBsAg/HBV-DNA negative; n ‫؍‬ 108) and anti-HCV antibody-positive (n ‫؍‬ 59) sera were identified as negative. The assay showed a detection limit of 4 ؋ 10 2 U/ml using recombinant antigen. Detection limits were compared in four serially diluted HBV high-titer sera. The HBcrAg assay demonstrated higher sensitivity than HBV-DNA transcription-mediated amplification ( Many hepatitis B virus (HBV) markers are used for diagnosing and monitoring hepatitis B patients. HBV-DNA tests, such as the branched-chain DNA (b-DNA) signal amplification assay (7, 31), and transcription-mediated amplification (TMA)-based (11) or PCR-based (12,14,20) assays are used to diagnose and monitor the efficacy of treatment. However, these methods require cumbersome procedures and expensive equipment, thus requiring considerable skill and high costs. These gene amplification assays also present some limitations (22,23,35). The b-DNA assay provides quantitative results but requires a long incubation time and lacks adequate sensitivity. Amplification assays have adequate sensitivity but are less quantitative.Immunoassays are generally easy and inexpensive. There have been a few reports of serum HBcAg assays with specimen pretreatment (4, 32). 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 is limited because of relatively low sensitivity and complex procedures.Serum HBeAg concentration reflects virus replication and hepatitis activity and is closely correlated with virus load in anti-HBe antibody-negative patients (8). Seroconversion of HBeAg to anti-HBe antibody reveals the inactive phase of infection (17,25). However, after seroconversion, many patients may exhibit reactivation and high viral load (3,10,18). In these cases, HBeAg is usually negative due to masking by anti-HBe antibody (24), although the HBeAg/anti-HBe immune complex can be indirectly detected according to the levels of alanine aminotransferase (ALT) and HBV-DNA (6). Therefore, HBcAg and HBeAg could be expected to be efficient markers of virus load if antibodies were inactivated and the antigens released.In the present study, for the purpose of developing a simple, sensitive, and inexpensive assay for determining HBV virus load, we targete...

Correlation between serum hepatitis B virus core‐related antigen and intrahepatic covalently closed circular DNA in chronic hepatitis B patients

“…Recently, a new assay was performed for the detection of hepatitis B core-related antigen (HBcrAg) consisting of HBV core antigen (HBcAg), HBeAg, and 22 kDa precore protein (p22cr) coded with precore/core gene [Kimura et al, 2002[Kimura et al, , 2005. p22cr is a precore protein from amino acid À28 to at least amino acid 150, containing an uncleaved signal sequence and lacking the C-terminal arginine-rich domain.…”

Patients with and without loss of hepatitis B virus DNA after hepatitis B e antigen seroconversion have different virological characteristics

“…Because the serum level of HBV core-related antigen mainly reflects that of HBe antigen [Kimura et al, 2002], the low level of HBV core-related antigen seen after seroconversion in both the inactive and active replication groups might have contributed to the occurrence of seroconversion. The pre-C and CP mutations, which were associated with the seroconversion, were frequent in the active replication group and rare in the inactive replication group, at least at around the time of seroconversion.…”

“…The HBVcrAg CLEIA measures the serum levels of HBe and HBc antigens simultaneously, using monoclonal antibodies, which recognize common epitopes of these two denatured antigens. However, the amount of HBV core-related antigen mainly reflects that of HBe antigen, because the concentration of HBe antigen in serum is much higher than that of HBc antigen [Kimura et al, 2002]. In the present study, the characteristic differences that may exist between patients with and without HBV DNA in serum after HBe antigen seroconversion were examined by comparing chronological changes of HBV DNA and HBV core-related antigen as well as by testing HBV genome mutations associated with the seroconversion.…”

“…The serum concentration of HBV core-related antigen was measured using the CLEIA reported previously [Kimura et al, 2002;Rokuhara et al, 2003]. In summary, 100 ml serum was mixed with 50 ml pretreatment solution containing 15% sodium dodecylsulfate and 2% Tween 60.…”

“…A chemiluminescence enzyme immunoassay (CLEIA) was developed previously for the detection of HBV corerelated antigen [Kimura et al, 2002;Rokuhara et al, 2003]. The HBV core-related antigen is expressed on HBe and core (HBc) antigens; both proteins are transcribed from the precore/core gene and their first 149 amino acids are identical.…”