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.

FIG. 1

The effect of pretreatment on the immunoreactivity of HCV core antigen (A) and the titer of anti-HCV core antibodies in anti-HCV antibody-positive sera (B) at various incubation temperatures. EIA results for anti-HCV core antibodies are means of duplicate assays, expressed as ratios of specimen absorbance to cutoff value (s/co ratios). Ratios over 1.0 are considered to show reactivity.

FIG. 2

Standard serum and recombinant HCV core antigen dilution curve. Standard serum (○) and recombinant HCV core antigen (●) were serially diluted four times with normal horse serum.

FIG. 3

(A) Gel filtration patterns of immunoreactive HCV core antigen in nonpretreated anti-HCV antibody-positive serum. Each fraction after fractionation on a Sepharose CL-4B column was pretreated and then measured by EIA. (B) Gel filtration patterns of immunoreactive HCV core antigen in pretreated anti-HCV antibody-positive serum on a Superdex 200 pg column. The total amount of HCV core antigen in each fraction after the chromatography of pretreated serum was measured by the EIA. The elution points for molecular markers are identified at the top. Vo, void volume; Vt, total column volume.

FIG. 4

Correlation between the concentrations of HCV core antigen and HCV RNA as detected by the AMPLICOR Monitor test. ○, negative sample as determined by the in-house RT-PCR; ●, sera with HCV of genotype 1; ▵, sera with HCV of genotype 2; ▴, sera with HCV of genotype 3; ×, sera with HCV of undetermined genotype; and □, sera for which HCV genotype was not tested.

FIG. 5

Patterns of various HCV markers in commercially available seroconversion panels. The data for anti-HCV antibody were obtained by Ortho EIA (3.0) by using the supplier’s data sheet. The concentrations of HCV core antigen (□) and HCV RNA as measured by AMPLICOR Monitor test (●) were measured at the points indicated. N, negative; P, positive.