摘要
为了提高对兔豆状囊尾蚴病的血清学诊断率,本研究用纯化的63 000囊液蛋白免疫BALB/c小鼠,获得了高效价一抗,再用63 000囊液蛋白作为包被抗原,以辣根过氧化物酶（HRP）标记的山羊抗鼠IgG为二抗,通过对各种试验条件的优化,建立了特异诊断兔豆状囊尾蚴病的阻断ELISA方法。结果显示,63 000囊液蛋白最佳包被浓度是5mg/L;一抗最佳稀释度为1∶800;最佳封闭时间是37℃3h;二抗最佳稀释度为1∶5 000,最佳反应时间是37℃30min;底物显色5min,终止反应后可先用目测法进行初步判定,并立即用酶标仪检测,根据D450值和判定标准确诊。用新建的阻断ELISA方法对兔的常见病,如兔脑炎原虫病、兔瘟、兔巴氏杆菌病、兔球虫病和弓形虫病进行了检测,均无交叉反应。从屠宰场和感染兔场随机采集150份血清样品,分别用阻断ELISA方法和间接血凝试验（IHA）进行比较检测,证明阻断ELISA方法的诊断率明显高于IHA。总之,阻断ELISA方法是一种简便、快速、灵敏和准确诊断兔豆状囊尾蚴病的新方法,适宜于基层兽医工作者应用和推广。
To enhance serological diagnostic rate for cysticercosis pisiformis,purified 63 000 cystic liquid proteins was used to inject BALB/c mouse immunologically and high titer primary antibody was obtained.The competitive ILISA method for peculiarly diagnosing cysticercosis pisiformis was established by serving purified 63 000 cystic liquid proteins as coating antigen,horse radish peroxidase-labeling goat anti-mouse IgG as secondary antibody and by mean of optimizing of various test condition.The results showed that optimal coating concentration of 63 000 cystic liquid proteins was 5 mg/L,optimal dilution of primary antibody was 1∶800,optimal blocking time was 37℃ at3 h,optimal dilution of secondary antibody was 1∶5 000,optimal reactive time was 37℃at30 min,coloring-development time of zymolyte was 5 min,after termination reaction the preliminary diagnosis was made by ocular estimate and according to D450 value and criterion of the definite diagnosis was made by microplate reader.Common diseases of rabbit,such as encephalitozoonosis,rabbit viral hemorrhagic disease,rabbit pasteurellosis,rabbit coccidiosis and toxoplasmosis,were detected by newly-built competitive ELISA method and cross reaction with cysticercosis pisiformis was not found in them.150 serous samples from rabbit abattoir and warren infected with cysticercosis pisiformis were examined by competitive ELISA method and indirect hemagglutination assay（IHA）and the results proved that diagnostic rate of competitive ELISA were higher than IHA.In short,competitive ELISA method is a simple,rapid,sensitive and accurate method for diagnosis of cysticercosis pisiformis and lends itself to application and popularity in grass-roots veterinarian.
作者
潘耀谦
冯春花
王帅
岳峰
王选年
刘兴友
PAN Yao-qian;FENG Chun-hua;WANG Shuan;YUE Feng;WANG Xiuan-nian;LIU Xieng-you(School of Life Science and Technology,Xinxiang Universtity,Xinxiang,Henan 453003,China;Xinxriang Animal Disease Prevention and Control Center,Xinxiang,Henan 453003,China;Department of Human Parasitology,School of Basic Medical Sciences,Xinxiang Medical University,Xinxiang,Henan 453003,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2018年第9期1704-1710,共7页
Chinese Journal of Veterinary Science
基金
河南省科技攻关基金资助项目(132102110118,142102110042)
关键词
豆状囊尾蚴病
阻断ELISA方法
囊液蛋白
间接血凝试验
血清学诊断
cysticercosis pisiformis
competitive ELISA method
cyst fluid protein
indirect hemagglutination test
serological diagnosis
