Sisi Luo^1,2, Bingyi Yang², Jiaoling Huang², You Wei², Zhixun Xie^{2 *}, Xiaofeng Li², Aiqiong Wu², Zhihua Ruan², Sheng Wang², Yanfang Zhang², Meng Li², Liji Xie², Ming Yan², Weiwei Wang¹and Ping Wei^{1 *}

¹Institute for Poultry Science and Health, College of Animal Science and Technology, Guangxi University, Nanning, Guangxi, China,

²Key Laboratory of China (Guangxi)-ASEAN Cross-Border Animal Disease Prevention and Control, Ministry of Agriculture and Rural Affairs, Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning, Guangxi, China

ABSTRACT  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.

KEYWORDS colloidal gold immunochromatography, fowl adenovirus serotype 4, monoclonal antibody, penton protein, rapid diagnosis

Frontiers in Veterinary Science. 2026 Mar 5:13:1758143. doi:10.3389/fvets.2026.1758143. eCollection 2026.