牛腺病毒3型免疫荧光检测方法的建立  

Establishment of immumofluorescence assay for detection of bovine adenovirus type 3

作　　者：贺倩 刘艳 郭敏 张平 司璐 李佳林 魏至栋 鱼轲 He Qian;Liu Yan;Guo Min;Zhang Ping;Si Lu;Li Jialin;Wei Zhidong;Yu Ke(Vaccine Division I,Lanzhou Institute of Biological Products Co.,Ltd.,Lanzhou 730046,China)

机构地区：[1]兰州生物制品研究所有限责任公司疫苗一室,兰州730046

出　　处：《国际生物制品学杂志》2022年第5期261-265,共5页International Journal of Biologicals

基　　金：国家科技支撑计划(2008BAI54B04-04)。

摘　　要：目的建立免疫荧光检测牛腺病毒3型(bovine adenovirus type3,BAV-3)的方法,用于生物制品用牛源材料的检测。方法分别以牛鼻甲骨细胞、传代牛肾细胞、原代牛肾细胞培养BAV-3,筛选最适宜的敏感细胞培养BAV-3,纯化获得抗原后免疫BALB/c小鼠。采用杂交瘤抗体技术制备抗BAV-3单克隆抗体(单抗),亲和层析法纯化后用异硫氰酸荧光素标记。用制备的单抗建立直接免疫荧光法检测BAV-3,并对方法的特异性、灵敏度及中间精密度进行验证。结果用抗BAV-3单抗检测牛腹泻病毒、副流感病毒3型、呼肠孤病毒、呼吸道合胞病毒、狂犬病毒、羊轮状病毒,均无交叉反应。采用该方法检测牛鼻甲骨细胞培养的BAV-3,3次传代可检出的最低病毒含量和标准差分别为0.10和0.05 CCID_(50)。结论建立的免疫荧光检测BAV-3方法具有良好的特异性和较高的灵敏度、中间精密度,可用于BAV-3的常规检测。Objective To develop an immunofluorescence method to detect bovine adenovirus type 3(BAV-3)in bovine-derived materials for biological products.Methods Bovine turbinate cells,Madin-Darby bovine kidney cells and primary bovine kidney cells were used to culture BAV-3,and the most sensitive cells were selected.BALB/c mice were immunized with purified BAV-3 antigen from culture.Using hybridoma antibody technology,anti-BAV-3 monoclonal antibody(McAb)was prepared,purified by affinity chromatography and labeled with fluorescein isothiocyanate,in order to develop direct immunofluorescence method to detect BAV-3.The specificity,sensitivity and intermediate precision were verified.Results The prepared anti-BAV-3 McAb did not cross react with bovine viral diarrhea virus,parainfluenza virus 3,reovirus,respiratory syncytial virus,rabies virus or lamb rotavirus.BAV-3 was cultured in bovine turbinate cells.The detection limit and standard deviation of BAV-3 after 3 passages were 0.10 and 0.05 CCID_(50),respectively.Conclusions The established immunofluorescence method for detecting BAV-3 has good specificity,high sensitivity,and good intermediate precision,whicn can be used for routine detection.

关 键 词：外源病毒 牛腺病毒3型 抗体 单克隆 免疫荧光试验 

分 类 号：R392-33[医药卫生—免疫学]