Development and diagnostic validation of a recombinant Hexon L1-based indirect enzyme-linked immunosorbent assay for detecting antibodies to fowl adenovirus serotype 8b in chickens.

Inclusion body hepatitis (IBH), caused by fowl adenoviruses (FAdVs), is a significant disease impacting poultry productivity. Recent data indicates that FAdV serotype 8b (FAdV-8b) is the predominant serotype associated with IBH cases in Thailand. Although the serum neutralization (SN) test is a commonly used method for detecting serotype-specific neutralizing antibodies, it has limitations in its routine application. This study developed and diagnostically validated an FAdV-8b-targeted indirect ELISA (iELISA) utilizing a recombinant protein of the FAdV-8b Hexon loop-1 region (rHexon8b), which was generated in E. coli and purified via Ni-NTA affinity chromatography. A total of 467 chicken sera with validated SN results were utilized for test optimization and diagnostic assessment. The optical density measured at 450 nm (OD₄₅₀) cut-off value was set at 0.112 and was supported by receiver operating characteristic analysis, which demonstrated excellent discriminatory performance with an area under the curve of 0.96 (95% confidence interval, 0.9407-0.9748; p < 0.0001). Utilizing the SN test as the reference standard, the rHexon8b iELISA demonstrated a diagnostic sensitivity of 92.73% and a diagnostic specificity of 89.33%, exhibiting substantial concordance between the two assays (Cohen's κ = 0.818). The assay showed no detectable cross-reactivity with the tested sera positive for major chicken pathogens or heterologous FAdV serotypes, including FAdV-2 and -11. The intra- and inter-assay coefficients of variation ranged from 0.92% to 6.80% and from 3.43% to 9.46%, respectively, indicating excellent repeatability and precision. These findings suggest that the rHexon8b iELISA is a rapid, reproducible, and practical serological screening tool for flock-level surveillance, vaccination monitoring, and epidemiological investigation of FAdV-8b-associated IBH in chickens.