Development of a colloidal gold immunochromatographic strip for the rapid detection of fowl adenovirus serotype 4 using prepared penton protein-specific monoclonal antibodies

Fowl adenovirus serotype 4 (FAdV-4) is the primary pathogen responsible for hydropericardium-hepatitis syndrome (HHS) and is associated with high mortality rates (20-80%) in 3-6-week-old chickens. This study aimed to develop a rapid and specific method for detecting FAdV-4. Two monoclonal antibodies (mAbs 6B3 and 8G11) against the FAdV-4 penton protein were successfully generated using hybridoma technology, both of which exhibited high titers (1:100,000) and strong serotype specificity. Specificity analysis confirmed that these mAbs recognized 12 FAdV-4 isolates from diverse origins without cross-reactivity to other FAdV serotypes or common avian pathogens. On this basis, a colloidal gold immunochromatographic assay was developed; systematic optimization of its key parameters yielded an optimal labeling pH of 8.3, an antibody labeling concentration of 7.2 μg/mL, and optimal coating concentrations for the test line (T-line) and control line (C-line) of 2.5 mg/mL and 1.5 mg/mL, respectively. Performance evaluation of the method demonstrated that it achieved a detection sensitivity of 7.81 × 10⁴ TCID₅₀/0.1 mL with a detection time of 15 min. Clinical sample validation revealed a positive concordance rate of 91.6%, a negative concordance rate of 100%, and an overall concordance rate of 98.6% relative to those of the ELISAs. The proposed colloidal gold immunochromatographic method offers advantages such as ease of operability, rapid visualization and high specificity and serves as a practical technical tool for onsite rapid diagnosis and epidemiological surveillance of FAdV-4 infection, thus it has high potential in the prevention and control of HHS.