牛副流感病毒3型双抗体夹心ELISA检测方法的建立  

Establishment and evaluation of a double antibody sandwich ELISA for detecting bovine parainfluenza virus type 3

作　　者：李丽阳[1,2] 李艳婷[1] 余丽芸[2] 侯喜林[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]黑龙江八一农垦大学生命学院,黑龙江大庆163319

出　　处：《中国预防兽医学报》2016年第2期132-136,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家科技支撑计划课题(2012BAD12B05);黑龙江省教育厅项目(12521372);黑龙江八一农垦大学校内培育课题资助计划项目(XZR2014-14);大庆市指导性计划项目(SZDFY-2015-42)

摘　　要：为建立牛副流感病毒3型（BPIV3）双抗体夹心ELISA（DAS-ELISA）检测方法,本研究采用纯化的病毒分别免疫小鼠与家兔,制备鼠抗BPIV3单克隆抗体（MAb）与兔抗BPIV3多克隆抗体（PAb）。以纯化的兔抗BPIV3 PAb作为包被抗体,鼠抗BPIV3 MAb作为检测抗体,采用棋盘滴定法确定DAS-ELISA的最适工作条件。结果显示,纯化的兔抗BPIV3 PAb与鼠抗BPIV3 MAb的效价分别为1：25 600和1∶12 800,均可与BPIV3病毒蛋白特异性结合。建立的DAS-ELISA方法捕获抗体最佳包被浓度为10μg/m L,37℃孵育1 h;检测抗体最佳工作浓度为2.5μg/m L,37℃孵育1 h;TMB室温显色30 min。判定的临界值为0.451。本研究建立的DAS-ELISA方法与牛轮状病毒、牛冠状病毒、牛传染性鼻气管炎病毒、牛呼吸道合胞体病毒均无交叉反应,特异性较强。检测下限为103TCID50。批间和批内变异系数均小于10%,具有良好的重复性。利用该方法和RT-PCR对75份牛鼻拭子临床样品进行检测,两者符合率为94.6%。本研究建立的检测BPIV3的DAS-ELISA方法适用于兽医基层临床样品的大规模检测。In this study,a double antibody sandwich enzyme-linked immunosorbent assay（DAS-ELISA） was developed for the detection of bovine parainfluenza virus type 3（BPIV3）.The polyclonal antibody and monoclonal antibody against the BPIV3 were firstly generated and used as the capture antibody and detection antibody,and then their optimal working concentrations in check-board titration were determined to be 10 μg/m L and 2.5 μg/m L,respectively.After the antigen-antibody complex was incubated at 37℃ for 1 h,the optical density at 450 nm was measured and the cut-off value was determined to be 0.451.The established DAS-ELISA could detect BPIV3 as low as 103TCID50,and exhibited no cross-reactivity with other virus,such as bovine rotavirus,bovine coronavirus,infectious bovine rhinotracheitis virus,bovine respiratory syncytial virus.Moreover,both inter-assay and intra-assay expressed as percentage of the coefficient of variation（CV） weren’t beyond 10%,demonstrating that DAS-ELISA had high sensitivity,specificity and repeatability.Lastly,DAS-ELISA was used to detect BPIV3 in 75 clinical cattle nasal swabs,and the results showed coincidence rate of 95% compared with RT-PCR method,suggesting that DAS-ELISA might be a convenient method for detecting BPIV3 in clinical surveillance.

关 键 词：牛副流感病毒3型 双抗体夹心ELISA 检测 

分 类 号：S852.65[农业科学—基础兽医学]