An optimized sandwich ELISA for quantitative detection of fowl adenovirus serotype 4 (FAdV-4) in chickens

Fowl adenovirus serotype 4 (FAdV-4) causes hydropericardium-hepatitis syndrome (HHS) in chickens, resulting in severe economic losses worldwide. Fast and accurate diagnostic methods for FAdV-4 are required to monitor its prevalence and provide plausible strategies for prevention. Sandwich ELISA is a common and rapid diagnostic technique; however, an FAdV-4 ELISA kit is not commercially available in China. Fiber 1 protein of FAdV-4 has been used as diagnostic targets. In this study, we firstly screened 5 monoclonal antibodies (mAbs) against fiber 1 from rabbits using phage display. ELISA and indirect fluorescence assay (IFA) showed these mAbs specifically bound to both fiber 1 and FAdV-4. Binding affinities of the mAbs forming optimal pair were lower than 10^-12 M, which recognized the knob and tail domain of fiber 1 by docking simulation, respectively. Next, sandwich ELISA for FAdV-4 was optimized, demonstrating excellent specificity, repeatability and reproducibility. The assay could detect as low as 0.532 ng/mL fiber 1 protein. For FAdV-4 diagnosis in clinical sera, the results of ELISA showed 97.44 % consistency with qPCR. Additionally, viral antigen levels in chick tissues determined by ELISA mirrored the viral loads by qPCR. The FAdV-4 kinetics in chicks quantified by ELISA and qPCR showed high consistency. In conclusion, we successfully developed a sensitive and specific ELISA for FAdV-4 diagnosis and quantification, offering a potential diagnostic tool in veterinary clinic.