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

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 g/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.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 transcriptionmediated 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 c...

Hepatitis B Virus DNA-negative Dane Particles Lack Core Protein but Contain a 22-kDa Precore Protein without C-terminal Arginine-rich Domain

“…Recombinant HBV Core-related Antigens-Recombinant HBcAg (rHBcAg, aa 1-183) and HBeAg (rHBeAg, aa Ϫ10 -149) were expressed and purified as described (21,22). The concentration of these antigens was determined using the BCA protein assay kit (Pierce) and bovine serum albumin standards according to the manufacturer's instructions.…”

“…We previously developed enzyme immunoassays (EIAs) for HBcAg (21) and HBV core-related antigens (HBcrAg) (22,23). Serum specimens were pretreated with SDS to release and denature antigens and to inactivate antibodies.…”

“…Serum specimens were pretreated with SDS to release and denature antigens and to inactivate antibodies. The HBcAg assay specifically measures core protein (21), and the HBcrAg assay measures precore/core proteins, including core protein and HBeAg (22,23).…”

Hepatitis B virus RNA is measurable in serum and can be a new marker for monitoring lamivudine therapy

“…HBV core and core-related antigen assay HBV core antigen (HBVcAg) and HBVcrAg in serum was measured using a chemiluminescence enzyme immunoassay as reported previously (14,15,18). In brief, 100 µL serum was mixed with pretreatment solution containing 15% sodium dodecylsulfate.…”

Detection and precipitation of hepatitis B core antigen using a fusion bacteriophage

“…Hence, the quantity of HBcAg would demonstrate the viral load as well as its genomic DNA. The concentration of HBcAg in serum is correlated with the level of HBV-associated DNA (Bredehorst et al, 1985;Kimura et al, 2003), meaning that HBcAg could be a marker for the viral load. Therefore, HBcAg has a Fig.…”