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 produc-tion, leading to huge economic losses worldwide. How-ever, there is no effective serological test for avian HEV. Researchers previously created a testing platform using the nanobody (Nb)-horseradish peroxidase (HO) fusion protein as an ultrasensitive probe to develop com-petitive ELISA (cELISA) to detect antibodies against different animal viruses. In this study, a rapid and reli-able 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 immu-nized 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, suggest -ing that the cELISA had good specificity. The coeffi-cients of variation were 0.91% to 4.21% (intra-assay) and 1.52% to 6.35% (inter-assay). Both cELISA and indirect ELISA 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 pro-tein, and the peptide (TFPS) in aa 601-604 was essen -tial for binding. The novel cELISA is a saving cost, rapid, useful, and reliable assay for the serological inves-tigation of avian HEV. More importantly, the peptide TFPS may be crucial to immunodominant antigen com-position and protection.