检测山羊贝氏柯克斯体抗体的间接ELISA方法的建立  

Establishment of an indirect ELISA for detecting antibodies against Coxiella burnetii in goats

作　　者：张小娟 钱沁清 张晓金 孙爱静[1] 俞樟森[1] 叶娜 贾广乐[3] ZHANG Xiaojuan;QIAN Qinqing;ZHANG Xiaojin;SUN Aijing;YU Zhangsen;YE Na;JIA Guangle(School of Medicine of Shaoxing University,Shaoxing 312000,China;School of Chemistry and Chemieal Engineering of Shaoxing University,Shaoxing 312000,China;Chinese Academy of Inspection and Quarantine,Beijing 100121,China)

机构地区：[1]绍兴文理学院医学院,浙江绍兴312000 [2]绍兴文理学院化学化工学院,浙江绍兴312000 [3]中国检验检疫科学研究院,北京100121

出　　处：《畜牧与兽医》2018年第12期47-52,共6页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金青年项目(51402195);绍兴文理学院校级科研项目(2015LG1002、2015LG1012)

摘　　要：以贝氏柯克斯体Ⅱ相全菌病原体沉淀作为抗原包被酶标板,建立检测山羊贝氏柯克斯体的间接ELISA (iELISA)方法,用该方法和美国爱德士贝氏柯克斯体检测试剂盒同时对445份临床山羊血清样品进行检测,评价二者的符合率。通过对i ELISA方法的关键反应条件进行优化,最终确定该方法的抗原包被浓度是0.5μg/mL,包被条件是4℃过夜;封闭液是10 g/L酪蛋白,封闭时间是45 min;血清稀释度是1∶400,反应时间是45 min;酶标二抗稀释度是1∶5 000,反应时间是30 min。经验证,建立的方法敏感性、特异性和重复性良好,与爱德士试剂盒的符合率为97.98%,证明该方法可用于山羊贝氏柯克斯体抗体的临床筛查。An indirect ELISA method for detecting antibody on Coxiella burnetii was established with the whole bacteria pathogen sediment of the C.burnetii attenuated strain phase Ⅱ as a coating antigen.Clinical goat serum samples(n=445) were simultaneously detected by this method and American IDEXX kits which was designed for detecting C.burnetii antibody,and the coincidence rate of two methods was evaluated.After optimizing the critical reaction conditions of the indirect ELISA,the optimal reaction conditions were obtained as follows:the concentration of the coating antigen was 0.5 ug/mL and the incubation condition was 4 ℃ overnight;the blocking buffer was 10 g/L casein solution and the blocking time was 45 min;the serum dilution was 1:400 and the reaction time was 30 min.The sensitivity,specificity and repeatability of this method were good and its coincidence rate with the IDEXX kit was 97.98%.Therefore,this indirect ELISA method could be applied to clinical screening of Coxiella burnetii antibody in goats.

关 键 词：山羊 贝氏柯克斯体 IELISA 

分 类 号：S852.62[农业科学—基础兽医学]