Development of a novel competitive ELISA based on nanobody-horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis E virus

Avian hepatitis E virus (avian HEV) increases poultry mortality and decreases egg production, leading to huge economic losses worldwide. However, there is no effective serological test for avian HEV. Researchers previously created a testing platform using the nanobody (Nb)-horseradish peroxidase (HRP) fusion protein as an ultrasensitive probe to develop competitive ELISA (cELISA) to detect antibodies against different animal viruses. In this study, a rapid and reliable cELISA was developed to test for antibodies against avian HEV using the same platform. Six anti-avian HEV capsid protein nanobodies were selected from an immunized Bactrian camel using phage display technology. The avian HEV-Nb49-HRP fusion protein was expressed and used as a probe for developing a cELISA assay to test for avian HEV antibodies. The cut-off value of the developed cELISA was 22.0%. There was no cross-reaction with other anti-avian virus antibodies, suggesting that the cELISA had good specificity. The coefficients of variation (CV) were 0.91-4.21% (intra-assay) and 1.52-6.35% (inter-assay). Both cELISA and indirect ELISA (iELISA) showed a consistency of 86.7% (kappa = 0.738) for clinical chicken serum samples, and coincidence between cELISA and Western blot was 96.0% (kappa = 0.919). The epitope recognized by Nb49 was located in aa 593-604 of the avian HEV capsid protein, and the peptide (TFPS) in aa 601-604 was essential for binding. The novel cELISA is a saving cost, rapid, useful and reliable assay for the serological investigation of avian HEV. More importantly, the peptide TFPS may be crucial to immunodominant antigen composition and protection.