Development of a Simple and Highly Sensitive Enzyme Immunoassay for Hepatitis C Virus Core Antigen

Abstract

A highly sensitive enzyme immunoassay (EIA) for the hepatitis C virus (HCV) core antigen (HCVcAg) was developed, and its performance was compared with that of the AMPLICOR HCV test (Roche Molecular Systems). The developed one-step pretreatment method, 30-min incubation of the specimen with a solution containing three different types of detergents (Triton X-100, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate [CHAPS], and sodium dodecyl sulfate), does not require any special device. Because the interfering anti-core antibody in the sample was sufficiently inactivated by the pretreatment, HCVcAg in the sample could be detected. The immunoreactivity on gel filtration was shifted from void fractions to those corresponding to the molecular mass range from 20 to 25 kDa, which is equal to the estimated molecular mass of HCVcAg, after the pretreatment. By the recovery test with HCVcAg-positive serum, the recovery rate was 93.5 to 106.5%. There was no interference with the EIA by anticoagulants or blood components in the serum. When the cutoff value was tentatively set at 0.5 mU/ml based on the distribution of healthy subjects’ sera, the sera of all healthy subjects (n = 125) and patients with hepatitis B (n = 50) were negative. HCVcAg was detected in sera from 57 of 73 individuals (78.1%) with anti-HCV antibody. Similarly, HCV RNA was detected in sera from 59 individuals (80.8%) with the AMPLICOR HCV as the qualitative test (AMPLICOR HCV test) and in sera from 54 individuals (74.0%) by the AMPLICOR HCV Monitor as the quantitative test (AMPLICOR Monitor test). Concentrations of HCVcAg and HCV RNA (measured by the AMPLICOR Monitor test) correlated significantly (r = 0.8, P < 0.001). On seroconversion panels, HCVcAg was detected during the early stage of infection, when anti-HCV antibodies had not been produced. This assay for HCVcAg is simpler than assays for HCV RNA based on gene technology and shows specificity and sensitivity equivalent to those of the AMPLICOR HCV test.

Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral‐related steatosis

“…Although ER HCV core is mainly attached to the cytosolic surface of the ER membrane, there is some indication that a minor fraction of core could also reach the ER lumen. In the present study, we were able to detect small amounts of core protein in the serum of core-expressing mice by enzyme immunoassay (40) in the absence of any liver cell necrosis (data not shown). One possibility is that the interaction of core with the lipids of the ER membrane (and perhaps intraluminal triglycerides) could hamper MTP-mediated transfer of triglycerides to the ER lumen and thus VLDL particle formation.…”

“…We would like to thank Lawrence Aggerbeck (Centre de Génétique Moléculaire-CNRS) for helpful discussions and providing anti-MTP and anti-PDI antibodies, Flora Zavala (INSERM U 25) for TNF-␣ and IL-6 level determinations, Shintaro Yagi (Advanced Life Science Institute Inc., Japan) for performing the serum core assay (40), Gérard Feldman and Marie-Elisabeth Samson-Brouma (INSERM U 327) for helpful discussions. G.P.…”

Repeated hepatocyte injury promotes hepatic tumorigenesis in hepatitis C virus transgenic mice

“…Total protein preparations of various tissues (brain, heart, lung, liver, kidney, intestine, spleen, pancreas and testis) from the transgenic mice were subjected to HCV core protein quantification assay using the sensitive EIA method. 19) Expression of the core protein was identified only in the liver and substantially lower levels were noted in other tissues. The levels of core protein expression in other tissues were similar to those observed in non-transgenic littermates (Fig.…”

“…Aliquots (100 µl) of these homogenates were assayed by the new EIA method described previously. 19) HCV core protein concentrations in the tissue samples were divided by total protein concentrations and expressed as fmol/g of total protein.…”

Monoclonal antibodies: Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus

“…They concluded that the Architect HCVAg assay is highly specific, easy to perform, and represents a valuable screening, diagnostic, and monitoring tool. Several investigators have demonstrated evidence of HCV antigens in liver tissue [3437] serum samples [38,39] and plasma samples [40] . We reported the detection of HCV NS4 antigen in the sera of infected HCV patients using the Dot ELISA technique as a rapid assay [41] .…”