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 x 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 (TMA) or HBeAg radio immunoassay (RIA) in the dilution test. HBcrAg concentrations correlated well with HBV-DNA TMA (r = 0.91, n = 29) and in-house real-time detection-PCR (r = 0.93, n = 47) in hepatitis B patients. On HBeAg/anti-HBe antibody seroconversion panels, the HBcrAg concentration changed in accordance with HBV-DNA levels. HBcrAg concentration provides a reflection of HBV virus load equivalent to HBV-DNA level, and the assay therefore offers a simple method for monitoring hepatitis B patients.

FIG. 1.

Standard curve and detection limit of HBcrAg assay. Recombinant ProHBe standard was diluted in normal human serum and detected by the HBcrAg assay. The assay reactivity was shown as relative luminescence intensity (RLI). (A) Standard curve; (B) detection limit. Results are means of 10 assays, and the error bars show 2 SD.

FIG. 2.

Dilution linearity of hepatitis B sera. Three HBV-DNA and HBeAg positive sera were serially diluted in normal human serum and detected by the HBcrAg assay.

FIG. 3.

Correlation between concentrations of HBcrAg and HBV-DNA in serum of hepatitis B patients. ⧫, HBeAg positive; ◊, anti-HBe positive; ▪, HBeAg and anti-HBe antibody negative. The HBV-DNA level was measured by TMA (detection limit, 3.7 to 8.7 LGE/ml) (A) or RTD-PCR (detection limit, 20 copies/ml) (B).

FIG. 4.

HBcrAg and HBV-DNA patterns in seroconversion panels. The data on HBsAg, HBeAg, and anti-HBs, anti-HBe, and anti-HBc antibodies were obtained from the supplier's data sheet. Closed symbols, positive; open symbols, under the cutoff. (A) BBI PHM935B panel. ▪ and □ HBcrAg [log(units/milliliter)]; ⧫ and ◊, HBV-DNA detected by the Roche Amplicor HBV Monitor test (detection limit, 4 × 10² to 4 × 10⁷ copies/ml); ▴ and ▵, in-house RTD-PCR (detection limit 20 copies/ml). (B) BCP HBV6281 panel. ▪ and □, HBcrAg [log (units/milliliter)]; ⧫ and ◊, HBV-DNA detected by BCP PCR (detection limit, 10² copies/ml).