Development and Application of a Nanobody-Based Competitive ELISA for Detecting Antibodies against Hepatitis E Virus from Humans and Domestic Animals

Hepatitis E virus (HEV) is a zoonotic pathogen that is widespread worldwide. At present, most enzyme-linked immunosorbent assay (ELISA) kits only detect antibodies against human HEV. In this study, a nanobody-horseradish peroxidase (HRP) fusion protein-based competitive ELISA (cELISA) with more convenience and spectral characteristics for HEV antibody detection was developed and used to detect HEV IgG in various species. First, 6 anti-swine HEV capsid protein nanobodies were screened using phage display technology from an immunized Bactrian camel. Then, HEV-Nb67-HRP fusions were expressed and used as a probe for developing a cELISA. The cutoff value of the cELISA was 17.8%, and there was no cross-reaction with other anti-swine virus antibodies, suggesting that the cELISA had good specificity. The intra-assay and interassay coefficients of variation (CVs) were 1.33 to 5.06% and 1.52 to 6.84%, respectively. The cELISA and Western blot showed a higher coincidence rate (97.14%, kappa value = 0.927) than cELISA and indirect ELISA (95.00%, kappa value = 0.876) in clinical swine serum samples. Finally, the seroprevalence of HEV IgG in humans, pigs, rabbits, cows, and goats was 30.67%, 19.26%, 8.75%, 27.59%, and 18.08%, respectively, suggesting that cELISA may have a broader scale for mammalian HEV antibody detection. These results suggest that the newly developed cELISA was rapid, low-cost, reliable, and useful for the serological evaluation of current HEV.IMPORTANCE HEV is thought to be a zoonotic infection and is widespread worldwide; it is beneficial to establish a more convenient and spectral method for HEV antibody detection. In this study, a convenient, time-saving, reproducible, highly sensitive, specific, and novel nanobody-based cELISA was developed and can be used to detect IgG antibodies against mammalian HEV. It provides a new technique for serological evaluation and ELISA-based diagnosis of HEV infection. HEV is thought to be a zoonotic infection and is widespread worldwide; it is beneficial to establish a more convenient and spectral method for HEV antibody detection. In this study, a convenient, time-saving, reproducible, highly sensitive, specific, and novel nanobody-based cELISA was developed and can be used to detect IgG antibodies against mammalian HEV. It provides a new technique for serological evaluation and ELISA-based diagnosis of HEV infection.