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.Hepatitis E virus (HEV), transmitted by the fecal-oral route, is the main cause of acute self-limiting hepatitis in many developing countries (1, 2, 19). Recently, HEV infection and the disease hepatitis E have also attained attention in industrialized countries (3,14). HEV is a single-stranded positive-sense RNA virus with a genome of approximately 7.2 kb. The viral genome contains three open reading frames (ORFs). There are four major genotypes of HEV identified, yet there is only a single serotype (19). In China, most sporadic cases are caused by genotype 4 (7, 13).Clinically, hepatitis E is not distinguishable from acute hepatitis caused by other viruses. Thus, the diagnosis of hepatitis E depends upon laboratory evidence of recent infection. Detection of HEV RNA in serum or feces by reverse transcription (RT) and nested or real-time PCR is direct evidence of acute infection; however, in addition to the expensiveness of the assays, viremia usually exists at the early acute phase and subsides soon after the apparent symptoms. Thus, the approach of detecting HEV RNA is not a practical way to clinically diagnose hepatitis E.Detection of IgM antibody against HEV (anti-HEV IgM) in serum can diagnose acute HEV infection. Commercial enzyme-linked immunosorbent assay (ELISA) kits for IgM are available; these assays were established based on recombinant antigens and/or synthetic polypeptides derived from ORF2 and/or ORF3 sequences (4,6,26,28). However...

Hepatitis E virus infection in Ghanaian blood donors – the importance of immunoassay selection and confirmation

“…Several studies have emphasized the unsatisfactory performance of anti‐HEV assays [30–40]. In this study, the performance of assays for the detection of anti‐HEV IgM and IgG from three manufacturers, Mikrogen (IgM and IgG assays), MP Biomedicals (IgM assay only) and Fortress Diagnostics (WanTai IgM and IgG assays), was compared.…”

“…In this study, the performance of assays for the detection of anti‐HEV IgM and IgG from three manufacturers, Mikrogen (IgM and IgG assays), MP Biomedicals (IgM assay only) and Fortress Diagnostics (WanTai IgM and IgG assays), was compared. Previous studies reported better sensitivity of the WanTai assays (Fortress Diagnostics) compared to other assays [36,37,40], but these had not been compared to the recom Well assays (Mikrogen) that differ from most other commercially available assays by utilizing capture antigens from both genotype 1 and genotype 3. Considering that no data were available regarding the HEV strains circulating in Ghana, the recom Well assays were favoured for this study.…”

“…Without testing serological assays on confirmed RNA‐positive sample from Ghana, it can only be speculated that the anti‐HEV IgM recom Well and MP Biomedicals assays lack specificity and/or the WanTai assay lacks sensitivity, at least for samples from sub‐Saharan countries as suggested by Pan et al. [40]. A lack of cross‐reactivity of the genotype 1 capture antigen in the WanTai assay with antibodies to genotype 3 is unlikely since it cross‐reacted well with antibodies to several genotype 3‐infected samples [35].…”

Restricted Enzooticity of Hepatitis E Virus Genotypes 1 to 4 in the United States

“…Moreover, in most assays, the antigens used for antibody capture are derived from HEV genotype 1 or genotypes 1 and 2. Because the HEV genotypes thus far identified to infect animals belong instead to genotype 3, genotype 4, and the newer putative genotypes, the possibility that low antibody detection rates reflect weak binding of antibody to heterotypic antigens rather than true antibody absence (2,19,24,44,45,48,62) cannot be excluded.…”

Development of a novel immunoperoxidase monolayer assay for detection of swine Hepatitis E virus antibodies based on stable cell lines expressing the ORF3 protein

“…ORF2 is located at the 3' end of the genome and encodes a capsid protein that encapsulates the viral RNA genome and contains an endoplasmic reticulum (ER) signal peptide and 3 putative N-glycosylation sites (Graff et al, 2008), which have been the focus of studies of antigenic sites (Ma et al, 2009;Pan et al, 2010). However, recent studies compared the polypeptide ends of the ORF1, ORF2 and ORF3 proteins.…”