A highly sensitive one-step nanobody-based immunoassay to specifically detect antibodies against fowl adenovirus serotype 4

Hepatitis-hydropericardium syndrome, caused by fowl adenovirus serotype 4 (FAdV-4), has resulted in significant economic damage to the poultry industry. To monitor viral exposure and vaccine efficacy, some traditional antibody-based immunoassays have been developed for detecting anti-FAdV-4 antibodies. However, these assays have some drawbacks including multi-step operations and higher production cost. Recently, nanobodies are regarded as a promising tool for developing immunoassays. In the study, 23 nanobodies against FAdV-4 were screened and expressed with horseradish peroxidase (HRP) in the HEK293T cells. Then, the FAdV-4-Nb28-HRP fusion protein was selected for developing competitive enzyme-linked immunoassays (cELISA) to detect anti-FAdV-4 antibodies in the chicken sera. The optimal concentrations and dilutions for the coating antigen, fusion protein and testing sera were determined to be 400 ng/well, 1:80 and 1:20, respectively. After the coated plates were vacuumized and stored, the operation of cELISA to detect clinical chicken sera was only one-step and the full time was 75 min. The cELISA also exhibited high sensitivity, specificity, reproducibility and good agreement with the commercial ELISA kit. When the sequential sera from the challenged chickens were tested, the cELISA showed superior sensitivity compared with the commercial ELISA kit. Moreover, epitope mapping revealed that the nanobody specifically recognized the sites GLN235 ASN236 SER238 of the fiber-1 protein, highly conserved among different FAdV-4 isolates and different from the FAdV-1 and -8. The results indicated that cELISA can specifically detect anti-FAdV-4 antibodies. Collectively, the developed one-step nanobody-based cELISA is an ideal method for epidemiological investigation and vaccine immune evaluation of FAdV-4.