Ponpukdee Nantawut, Wangman Pradit, Sopa Chatchapong, Chaivisuthangkura Parin, Longyant Siwaporn
Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, Thailand.
Center of Excellence in Animal, Plant and Parasite Biotechnology, Srinakharinwirot University, Bangkok, Thailand.
J Fish Dis. 2025 Aug 11:e70041. doi: 10.1111/jfd.70041.
The global tilapia farming industry faces a significant threat from a widespread pandemic caused by tilapia lake virus (TiLV), which can cause mortality rates of up to 90% within 14 days in infected fish. This study describes the generation, evaluation and comparison of monoclonal antibodies (Mabs) with molecular techniques for TiLV detection. The antigen was produced as a recombinant protein using a bacterial expression system, specifically targeting segment 8 of the TiLV genome. Four Mabs successfully detected TiLV-infected tilapia samples using dot blotting and Western blotting assays, with detection limits of 0.78 ng/spot for each Mab. Notably, a combination of Mabs enhanced detection sensitivity fourfold and exhibited no cross-reactivity with bacterial and viral pathogens in tilapia. Furthermore, two Mabs effectively localised TiLV within infected tilapia tissue via immunohistochemistry. Although immunoassay-based techniques demonstrated high specificity, their sensitivity was approximately thirtyfold lower than that of semi-nested RT-PCR. Nonetheless, the Mabs accurately detected TiLV in all 20 confirmed positive samples via dot blotting and offer advantages in terms of reduced processing time and cost per reaction. These Mabs will contribute to future efforts aimed at preventing and controlling the spread of tilapia lake virus disease in aquaculture by facilitating the rapid identification and diagnosis of TiLV-infected fish.
全球罗非鱼养殖业面临着由罗非鱼湖病毒（TiLV）引发的广泛疫情带来的重大威胁，这种病毒可使受感染的罗非鱼在14天内死亡率高达90%。本研究描述了用于TiLV检测的单克隆抗体（Mab）的产生、评估及与分子技术的比较。抗原是使用细菌表达系统作为重组蛋白产生的，专门针对TiLV基因组的第8节段。四种单克隆抗体通过斑点印迹法和蛋白质印迹法成功检测出TiLV感染的罗非鱼样本，每种单克隆抗体的检测限为0.78 ng/斑点。值得注意的是，单克隆抗体的组合使检测灵敏度提高了四倍，并且与罗非鱼中的细菌和病毒病原体无交叉反应。此外，两种单克隆抗体通过免疫组织化学有效地在受感染的罗非鱼组织中定位了TiLV。尽管基于免疫测定的技术显示出高特异性，但其灵敏度比半巢式RT-PCR低约30倍。尽管如此，单克隆抗体通过斑点印迹法在所有20个确诊阳性样本中准确检测出TiLV，并且在减少处理时间和每个反应成本方面具有优势。这些单克隆抗体将有助于未来通过促进快速鉴定和诊断TiLV感染的鱼类来预防和控制罗非鱼湖病毒病在水产养殖中的传播。
