摘要
为建立检测细胞培养物和组织切片中羊口疮病毒(Orf virus,ORFV)的间接免疫荧光(IFA)方法,用已知滴度的ORFV感染牛睾丸上皮细胞,羊口疮病毒单克隆抗体(anti-ORFV mAb)为一抗,荧光素标记羊抗小鼠IgG为二抗,通过反应条件的优化,建立细胞培养物ORFV的IFA检测方法,并制备ORFV感染的山羊唇部组织冰冻切片,对组织中病毒进行检测。结果显示,用牛睾丸上皮细胞增殖的羊口疮病毒TCID 50达到10-6.29/0.1 mL,ORFV适宜的接种滴度和培养条件为103.70 TCID 50 ORFV接种细胞,37℃、体积分数为5%的CO 2恒温箱培养42 h,荧光素标记羊抗小鼠二抗适宜工作浓度为1∶400稀释,此时羊口疮病毒单抗应用于IFA检测的特异性和灵敏度最佳,且可应用于病料组织中ORFV的定位检测。表明成功建立了羊口疮病毒单抗检测牛睾丸上皮细胞和组织切片中ORFV的间接免疫荧光方法,有助于ORFV感染的实验室诊断及其在感染细胞中的定位和动态分布研究。
To establish the indirect immunofluorescence assay(IFA)for detecting ORFV in cell cultures and tissue sections,bovine testicular epithelial cells were infected with ORFV of known titers,following by using the anti-ORFV mAb as the primary antibody and fluorescein-labeled goat anti-mouse IgG as the second antibody.The initial infection titer and time of IFA method for detection of ORFV in cell cultures were explored and optimized.Frozen sections of ORFV-infected goat lip tissue were prepared for detection of virus in tissues.The results showed that the TCID 50 of ORFV proliferated by bovine testicular epithelial cells reached 10-6.29/0.1 mL,the appropriate inoculation titer of ORFV and culture conditions were that cells were vaccinated with 103.70 TCID 50 ORFV and cultured for 42 h at incubator(37℃5%CO 2)The suitable working concentration of fluorescein-labeled anti-mouse secondary antibody was 1∶400.At this time,the specificity and sensitivity of anti-ORFV mAb for IFA is optimal,and it can be applied to the localization detection of ORFV in the infected tissue.It indicated that the indirect immunofluorescence method of ORFV in bovine testicular epithelial cells and tissue sections were successfully established,which will contribute to the laboratory diagnosis of ORFV infection and its localization and dynamic distribution in infected cells.
作者
宁梦夏
程红玉
马文涛
陈德坤
NING Meng-xia;CHENG Hong-yu;MA Wen-tao;CHEN De-kun(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China)
出处
《动物医学进展》
北大核心
2020年第4期42-47,共6页
Progress In Veterinary Medicine
基金
咸阳市重大科技项目(2017K01-34)
青海省重点研发与转化计划项目(2018-NK-125)。
关键词
羊口疮病毒
单克隆抗体
间接免疫荧光
ORFV
monoclonal antibody
indirect immunofluorescence assay
