Quantitation of immunoglobulin to hepatitis E virus by enzyme immunoassay

Abstract

We developed a quantitative enzyme immunoassay (EIA) for antibody to hepatitis E virus (HEV) by using truncated HEV capsid protein expressed in the baculovirus system to improve seroepidemiology, to contribute to hepatitis E diagnosis, and to enable vaccine evaluations. Five antigen lots were characterized; we used a reference antiserum to standardize antigen potency. We defined Walter Reed antibody units (WR U) with a reference antiserum by using the four-parameter logistic model, established other reference pools as assay standards, and determined the conversion factor: 1 WR U/ml = 0.125 World Health Organization unit (WHO U) per ml. The EIA performed consistently; median intra- and inter-test coefficients of variation were 9 and 12%, respectively. The accurate minimum detection limit with serum diluted 1:1,000 was 5.6 WR U/ml; the test could detect reliably a fourfold antibody change. In six people followed from health to onset of hepatitis E, the geometric mean antibody level rose from 7.1 WR U/ml to 1,924.6 WR U/ml. We used the presence of 56- and 180-kDa bands by Western blotting as a confirmatory test and to define true-negative and -positive serum specimens. A receiver-operating characteristics plot identified 30 WR U/ml as an optimum cut-point (sensitivity, 86%; specificity, 89%). The EIA detected antibody more sensitively than a commercially available test. The EIA was transferred to another laboratory, where four operators matched reference laboratory results for a panel of unknowns. Quantitation of antibody to HEV and confirmation of its specificity by Western blotting make HEV serology more meaningful.

FIG. 1.

SDS-PAGE analysis of five rHEV antigens lots stained with colloidal Coomassie blue. Lot 4b was 62-kDa antigen, and all other lots were 56-kDa antigen.

FIG. 2.

Western blots of two rHEV antigen lots run with Sf9 host cell antigens (7 μg of each sample, denatured without mercaptoethanol); blots are probed with normal human control antiserum (A), pool 1 human antiserum to HEV (B), monoclonal antibody to HEV capsid (C), and antiserum to Sf9 cells infected with baculovirus (D).

FIG. 3.

Parallel line assay comparing three reference antibodies: the WHO reference standard (Std.) (dashed line), pool 4 (dotted line), and pool 1 (solid line).

FIG. 4.

Plots of the HRP label (TAG), midrange positive control (Pool 5), negative control (Neg), and no-serum control (Antigen) over 50 technically adequate assays.

FIG. 5.

Levels of antibody to HEV in six patients with hepatitis E determined before infection, when acutely ill (vertical reference line at day 0), and during convalescence. The lower horizontal reference line is the more specific assay cut-point (40 WR U/ml), and the upper reference line is the geometric mean level of acute illness antibody (1,925 WR U/ml).

FIG. 6.

Histogram of EIA values in specimen sets from donors lacking plausible exposure to HEV. The top panel includes 178 infants and children from Bangkok, Thailand (mean = 3.6 WR U/ml, SD = 3.1). The bottom panel includes 360 adults from the United States (mean = 7.1 WR U/ml, SD = 6.1).

FIG. 7.

Western blot confirmation test. Lanes: 1, pool 5 positive control; 2, negative control; 3, negative specimen with 30 WR U/ml; 4, positive specimen with 56 WR U/ml; 5, positive specimen with 137 WR U/ml; 6, positive specimen with 713 WR U/ml.

FIG. 8.

Receiver-operating characteristics plot for EIA based on true-negative and -positive samples defined by Western blotting.

FIG. 9.

Comparison of antibody quantitation by WRAIR and Genelabs Technologies EIA. The dashed horizontal reference line is the cut-point for the Genelabs test.