羊源肺炎克雷伯菌间接免疫荧光检测方法的建立

Establishment of indirect immunofluorescence assay for Klebsiella pneumoniae

将羊肺脏中分离鉴定后的肺炎克雷伯菌制成菌体免疫抗原,免疫健康家兔后获得兔抗羊源肺炎克雷伯菌多克隆抗体。依据此多克隆抗体建立检测肺炎克雷伯菌的间接免疫荧光检测方法,并对检测条件进行了筛选和优化。结果表明,多克隆抗体稀释度为1∶80,FITC标记羊抗兔IgG稀释度为1∶400,孵育时间为30 min时,羊源肺炎克雷伯菌的特异性黄绿色荧光最清晰,非特异性荧光最弱。并通过临床检测证明该方法具有简便、特异性高的优点,为羊源肺炎克雷伯菌的流行病学、血清学调查和临床诊断奠定基础。

Klebsiella pneumoniae(K.pneumoniae)can cause disease in humans and a variety of animals under certain conditions,but there is no relevant research on the detection method of Klebsiella pneumoniae of sheep origin.In this study,a polyclonal antibody against Klebsiella pneumoniae was obtained by immunizing healthy rabbits with Klebsiella pneumoniae isolated from lungs of sheep.An indirect immunofluorescence assay for detecting Klebsiella pneumoniae was established based on this polyclonal antibody,and the assay conditions were optimized.The results showed that under the conditions of 80 times dilution of polyclonal antibody,400 times dilution of FITC-labeled goat anti-rabbit IgG and incubation for 30 min,the specific yellow-green fluorescence of Klebsiella pneumoniae was the clearest and the non-specific fluorescence was the weakest.Furthermore,the method was proved to be simple and specific by clinical tests,which laid a foundation for the epidemiological and serological investigation and clinical diagnosis of Klebsiella pneu moniae.