Yin Kequan, Ren Jingwei, Zhu Yue, Xu Lijuan, Yin Chao, Li Yang, Yuan Yu, Li Qiuchun, Jiao Xinan
Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China, Yangzhou University, Yangzhou, China.
Jiangsu Key Lab of Zoonosis/Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China.
Front Vet Sci. 2019 Nov 5;6:386. doi: 10.3389/fvets.2019.00386. eCollection 2019.
Pullorum disease remains an epidemic in the poultry industry in China. The causing pathogen is a host-restricted serovar Pullorum, which can spread through both horizontal and vertical transmissions. To eradicate the pullorum disease from poultry farms, it is necessary to specifically monitor the prevalence of the bacterial infection in adult chicks. In this study, we constructed a new competitive ELISA method based on the development of monoclonal antibodies (MAbs) against a specific immunogen of . Pullorum, IpaJ protein. In total, eight MAbs against IpaJ were prepared using the purified recombinant His-IpaJ protein as the immunogen. Characterization of the eight MAbs demonstrated that 4G5 can be used as the competitive antibody in ELISA. A competitive ELISA was subsequently developed using purified MBP-IpaJ as the capture (0.5 μg/ml) and the HRP-labeled 4G5 (0.14 μg/ml) as the competitive antibody, respectively. A specificity test demonstrated that the ELISA assay can differentiate antisera of . Pullorum-infected chickens from that of . Gallinarum and . Enteritidis. Furthermore, 4 out of 200 clinical antisera collected from a poultry farm were detected to be . Pulloram positive using this method. The plate agglutination test (PAT) and the previously established indirect ELISA confirmed that these positive antisera reacted specifically with . Pullorum. We propose that the established competitive ELISA assay based on MAb against IpaJ protein, is a novel and quick method that can detect . Pullroum infection in the poultry industry.
鸡白痢在中国家禽业中仍是一种流行病。致病病原体是宿主限制性血清型鸡白痢沙门氏菌，它可通过水平和垂直传播。为了在家禽养殖场根除鸡白痢，有必要专门监测成年雏鸡中细菌感染的流行情况。在本研究中，我们基于针对鸡白痢沙门氏菌的一种特异性免疫原IpaJ蛋白开发单克隆抗体构建了一种新的竞争ELISA方法。总共使用纯化的重组His-IpaJ蛋白作为免疫原制备了8种针对IpaJ的单克隆抗体。对这8种单克隆抗体的表征表明4G5可作为ELISA中的竞争抗体。随后开发了一种竞争ELISA，分别使用纯化的MBP-IpaJ作为捕获抗体（0.5μg/ml）和HRP标记的4G5（0.14μg/ml）作为竞争抗体。特异性试验表明该ELISA检测方法能够区分感染鸡白痢沙门氏菌的鸡血清与感染鸡伤寒沙门氏菌和肠炎沙门氏菌的鸡血清。此外，使用该方法从一个家禽养殖场收集的200份临床血清中有4份被检测为鸡白痢阳性。平板凝集试验（PAT）和先前建立的间接ELISA证实这些阳性血清与鸡白痢沙门氏菌发生特异性反应。我们认为基于针对IpaJ蛋白的单克隆抗体建立的竞争ELISA检测方法是一种可检测家禽业中鸡白痢感染的新颖且快速的方法。
