禽波氏杆菌免疫荧光检测和抗体检测ELISA方法的建立与应用  

Establishment and Application of Immunofluorescence Assay and Indirect ELISA for Detection of B.avium

作　　者：杨春晓[1] 王小娥[1] 刘冠华[1] 肖海君[1] 崔金生[1] 朱瑞良[1] 

机构地区：[1]山东农业大学动物科技学院,泰安271018

出　　处：《畜牧兽医学报》2008年第12期1715-1720,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(3067011430740077);山东省自然科学基金项目(Y2006D09)

摘　　要：采用改良的Wooldridge法提取了禽波氏杆菌外膜蛋白(OMP),通过Bradford方法测定禽波氏杆菌OMP含量为320μg/mL,SDS-PAGE电泳检测发现禽波氏杆菌P5株OMP含有5种成分,相对分子质量分别为58、47、41、36、24 ku;P8株OMP含有6种成分,相对分子质量分别为58、47、41、38、211、6 ku。将提取的禽波氏杆菌OMP免疫健康青紫兰兔,每周免疫1次,共免疫4次,获得了兔抗禽波氏杆菌OMP高免血清,以此高免血清建立了检测禽波氏杆菌的间接ELISA和间接免疫荧光(IFA)方法,并对其工作条件进行了优化和筛选,试验结果表明间接ELISA抗原、抗血清最佳工作浓度分别为10μg/mL、1∶12 800,酶标二抗工作浓度为1∶5 000,最佳包被条件为4℃2、4 h,最佳封闭条件为37℃1、h;间接免疫荧光(IFA)兔高免血清释释度为1∶20,FITC标记羊抗兔IgG稀释度为1∶10,感作时间为45 min时,禽波氏杆菌特异性黄绿色荧光最清晰,非特异性荧光最弱。经临床检测证明该两种方法均具有简便、快速、特异性强的特点,为禽波氏杆菌病的流行病学、血清学调查和临床快速诊断奠定了基础。In this study, out membrane proteins of Bordetella avium strains P5 and P8 were prepared by improved Wooldridge method. The contents of Bordetella avium OMP were determined by Bradford method. SDS-PAGE electrophoresis was performed to identify Bordetella avium OMP. Prepared OMP immunogen was immunized to rabbits for preparation of high titer immuno serum. And then detections of indirect ELISA and indirect immunofluorescence were established by optimization and screening of conditions. The result showed that the contents of Bordetella avium OMP was 320 μg/mL. The protein molecular weight of OMP are 58,47,41,36,24 kDa in P5, and are 58,47,41,38,21,16 kDa in P8. The detection technique of I-ELISA and IFA were simple, rapid and specific. They can be used in study of epidemiology and serology of Bordetella avium. The two methods laid a foundation for fast diagnosis of Bordetella avium.

关 键 词：禽波氏杆菌 OMP 间接ELISA 间接免疫荧光(IFA) 

分 类 号：S852.61[农业科学—基础兽医学] S855.12[农业科学—兽医学]