Double-Antigen Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis E Virus-Specific Antibodies in Human or Swine Sera

Abstract

A new double-antigen sandwich-based enzyme-linked immunosorbent assay (ELISA) for the detection of total antibodies (immunoglobulin G [IgG] and IgM) specific for hepatitis E virus (HEV) was developed by utilizing well-characterized recombinant protein ET2.1 and its peroxidase-labeled counterpart. Our study showed that the ELISA detected all the positive patient samples (n ‫؍‬ 265) regardless of whether they contained IgM or IgG antibodies, or both, while it maintained an excellent specificity of 98.8% with samples from various patient or healthy control groups (total number of samples, 424). The test had a detection limit for anti-HEV IgG antibodies that was equivalent to 62 mIU/ml of the international reference. Compared with the serological status of the specimens determined on the basis of tests performed at the individual collection sites, the testing outcome generated by the new ELISA had a good agreement of 99.3%, with a kappa value of 0.985. The positive predictive value and the negative predictive value for the new test reached 98.1% and 100%, respectively. This ELISA had a positive delta value of 4.836 and a negative delta value of 3.314 (where delta is a measure of the number of standard deviations by which the cutoff is separated from the mean of the sample groups) (N. Crofts, W. Maskill, and I. D. Gust, J. Virol. Methods 22:51-59, 1988), indicating that it had an excellent ability to differentiate the infected and noninfected cohorts. Furthermore, the new design enables the detection of antibodies not only in human samples but also in pig samples. Our preliminary data showed that the ELISA could detect seroconversion in samples from pigs at as early as 14 days postinoculation. The potential utility of detecting specific antibodies in pigs will be an added advantage for managing the disease, with suggested zoonotic implications.

Seroprevalence study in forestry workers from eastern Germany using novel genotype 3- and rat hepatitis E virus-specific immunoglobulin G ELISAs

“…Due to failure in expression of the entire ORF2 in the E. coli expression system, carboxy-and amino-terminal segments of the CP were generated in E. coli. In line with previous investigations [40][41][42], the carboxy-terminal segments spanning aa residues 326-608 and 326-660 were found to be highly reactive with a pool of anti-HEV positive human sera. Additionally, immunodominant antigenic regions were previously found in this region using peptide-based assays [43,44].…”

Good Performance of Immunoglobulin M Assays in Diagnosing Genotype 3 Hepatitis E Virus Infections

“…HEV IgM ELISA 3.0 and the Assure HEV IgM rapid test (MP Diagnostics) are based on a conformational ORF2-encoded epitope from a genotype 1 strain, the ORF2.1 antigen, considered highly conserved between HEV strains (1). Their good sensitivities observed in the present work are in keeping with a previous study that demonstrated that the ORF2.1 antigen is suitable for detecting total anti-HEV antibodies in swine infected with genotype 3 (5).…”

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

“…First, it incorporates antigens representing all four HEV genotypes, so allowing for the detection of antibodies generated in the host regardless of the genotype of the infecting HEV. Other DASAs developed for anti-HEV detection (14,22,55,63) mostly utilize antigenic preparations derived from one genotype only. Second, the highly immunodominant p166 antigens (37) are used to capture antibody, as well as to report its solid-phase capture, thereby conferring both sensitivity and specificity to anti-HEV detection.…”