Abstract
Hepatitis E virus (HEV) infects both humans and animals, with an overall human mortality rate generally less than 1%, but as high as 20% among pregnant women. HEV strains fall into 4 major genotypes. Zoonotic genotypes 3 and 4 associate with sporadic human and animal HEV cases in many industrialized countries. To date, collective evidence implicates pigs as the main HEV reservoir, justifying the importance of monitoring HEV infection rates in pig herds to prevent human illness. Due to the lack of a robust in vitro cell culture system for viral propagation, no “gold standard” assay has yet been developed to detect HEV infection in domestic pigs. 1E4, a monoclonal antibody (mAb) specific for the C-terminal 268 amino acids of HEV genotype 4 ORF2 capsid protein (sORF2-C), was generated and conjugated to horseradish peroxidase (HRP) for use in a blocking ELISA (bELISA). Optimal sORF2-C coating antigen concentration (8 μg/ml), HRP-1E4 dilution (1:1000), and test pig serum dilution (1:20) were determined using a checkerboard titration test. A cut-off value of 16.9% was chosen to differentiate between positive vs. negative sera after mean percent inhibition (PI) testing of 230 negative pig sera. Compared with the indirect ELISA (iELISA), western blot, and a commercial ELISA kit for detecting anti-HEV antibodies in human sera, the bELISA showed no statistical differences and statistically high coincidence of 93.23%, 92%, and 95% with the other tests, respectively. A blocking ELISA (bELISA) for detecting anti-HEV antibodies in pig serum samples was developed with high sensitivity and high specificity comparable to that of the indirect ELISA. The bELISA results exhibited high agreement with iELISA, western blot, and a commercial ELISA kit designed to detect human anti-HEV antibodies. Therefore, bELISA should serve as an ideal method for large-scale serological investigation of anti-HEV antibodies in domestic pigs.
Highlights
Hepatitis E virus (HEV) can cause self-limiting hepatitis in humans that is a serious public health problem in many developing countries, as well as some developed countries [1]
For serological diagnosis in pigs, anti-HEV antibodies are universally detected using two methodologies: commercial kits optimized for detection of human anti-HEV antibodies that have been adapted to use for swine and inhouse indirect enzyme-linked immunosorbent assays using genotype 3 or 4 HEV ORF2 proteins as coating antigens [24,25,26]
The 45 serum samples collected 0 to 56 dpi from the 5 pigs challenged with CHN-SDsHEV were used to block binding of the three horseradish peroxidase (HRP)-monoclonal antibody (mAb) to sORF2-C protein bound to a solid phase
Summary
Hepatitis E virus (HEV) can cause self-limiting hepatitis in humans that is a serious public health problem in many developing countries, as well as some developed countries [1]. For serological diagnosis in pigs, anti-HEV antibodies are universally detected using two methodologies: commercial kits optimized for detection of human anti-HEV antibodies that have been adapted to use for swine and inhouse indirect enzyme-linked immunosorbent assays (iELISAs) using genotype 3 or 4 HEV ORF2 proteins as coating antigens [24,25,26]. These iELISAs have not yet been validated, due to the absence of appropriate “gold standard” for comparison [1] and studies have shown that iELISAs often provide discordant results and non-specific background signals [27]. On the basis of these findings, the bELISA should serve as a useful method for large-scale serological investigation of anti-HEV antibodies in pigs
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