Application of truncated immunodominant polypeptide from hepatitis E virus (HEV) ORF2 in an assay to exclude nonspecific binding in detecting anti-HEV immunoglobulin M

Abstract

The diagnosis of recent hepatitis E virus (HEV) infection depends on serologic testing for anti-HEV IgM; however, false-positive results may occur. In the present study, we cloned the ORF2 fragment of genotype 4 HEV and demonstrated that a subregion covering amino acids 459 to 607 in ORF2 forms the immunodominant B-cell epitopes, as it does in genotype 1 viruses. Truncation of several residues from either the N or C terminus of the polypeptide abolished the reactivity of anti-HEV from naturally infected persons. By the combination of high reactivity of the immunodominant polypeptide and poor reactivity of the truncated polypeptide, we established an indirect enzyme-linked immunosorbent assay (ELISA) to detect anti-HEV IgM. In this assay, all 37 sera that were HEV RNA positive reacted with the immunodominant polypeptide but not with the truncated one, and none of 159 sera from healthy persons reacted with either of the polypeptides. In retesting of 117 sera that originally tested positive for anti-HEV IgM, using a Genelabs kit, only 34 were positive and 83 were negative. Western blot analyses and other experiments strongly indicated that these 83 discordant sera were negative for anti-HEV IgM. Furthermore, among the 117 sera, 5 reacted with both the immunodominant and truncated polypeptides, with comparable optical densities at 450 nm. However, their reactivity was demonstrated to result from nonspecific binding. Together, the data indicate that the poor reactivity of a truncated ORF2 polypeptide can be used to exclude nonspecific binding in the detection of anti-HEV IgM.

FIG. 1.

Expression and purification of histidine-tagged HEV ORF2 polypeptides in E. coli. (A) SDS-PAGE of polypeptides spanning amino acids 459 to 607, 472 to 607, and 459 to 594. 0 h and 4 h, 0 and 4 h after adding IPTG (isopropyl-β-d-thiogalactopyranoside). (B) Standard SDS-PAGE of purified polypeptides. (C) SDS-PAGE of purified polypeptides, in Laemmli buffer, which were not heated.

FIG. 2.

Antigenic characterization of ORF2 polypeptides by Western blot analysis. His₆-tagged polypeptides in Laemmli buffer were not heated before being run in the gel. Lanes 1 to 3 indicate the polypeptides covering amino acids 459 to 607, 472 to 607, and 459 to 594 in ORF2, respectively. The color was developed by the DAB enhanced liquid substrate system. (A) The membrane was probed with peroxidase-labeled anti-His₆. (B and C) The membranes were incubated with the serum from a hepatitis E patient and then probed with peroxidase-labeled anti-human IgG (B) and IgM (C).

FIG. 3.

Western blot analysis of eight sera that reacted with both immunodominant and truncated ORF2 polypeptides. His₆-tagged polypeptides in Laemmli buffer were not heated before being run in the gel. Lanes 1 and 2, histidine-tagged polypeptides covering aa 459 to 607 and aa 472 to 607 in ORF2, respectively. Anti-His₆, the membranes were probed with peroxidase-labeled anti-His₆; E04 to E90, the membranes were incubated with each of the sera and then probed with peroxidase-labeled anti-human IgM. The color was developed by the DAB enhanced liquid substrate system.