摘要
为建立阿卡斑病毒抗体检测方法,本研究以纯化的阿卡斑病毒作为包被抗原,以制备的抗阿卡斑病毒的多克隆抗体与待检血清竞争结合抗原,采用方阵滴定法优化反应条件,建立了阿卡斑病毒竞争ELISA(cELISA)抗体检测方法。通过对60份牛、羊阿卡斑病毒抗体阳性血清和40份阴性血清样品的检测,确定阴阳性样品抑制率(PI)临界值为60%;特异性试验结果显示,该方法对BTV-1、BTV-4、BTV-9、BTV-16、小反刍兽疫、中山病病毒、鹿流行性出血热病毒(EHDV)、羊痘和O型、A型、Asia-1型口蹄疫病毒阳性血清无交叉反应;敏感性试验结果显示,标准阳性血清1颐128倍稀释时,检测结果仍为阳性,SNT最低检出为,敏感性高于微量血清中和试验(SNT)检测结果(1颐64);批内和批间变异系数(CV)在0.54%~5.27%和0.3%~9.3%之间,具有较好的可重复性;用该方法与法国IDVET公司的同类试剂盒对85份牛、羊血清样品同时检测,结果显示二者符合率为91.76%,可以替代进口同类产品。本研究建立的检测方法特异性强、敏感性高、重复性好,可以用于阿卡斑病毒抗体检测和流行病学调查。
In order to established a assay for detection of the antibody against Akabane virus,a competitive ELISA(cELISA)was developed with the specific polyclonal competing antibodies prepared by immunization of rabbits with the Akabane virus isolated from Yunnan province.A total of 60 negative antibody samples and 40 positive antibody samples of Akabane virus were detected by the established cELISA,and the cut-off value of the present inhabitation was 60%.The detection results showed that the cELISA was specific for Akabane virus detection,and no crossing-reaction was found to the positive sera of BTV-1,BTV-4,BTV-9,BTV-16,PPRV,Chuzan virus,EHDV,FMV-O,FMDV-A and FMDV-Asia1.The sensitivity test results shown that the established assay was able to detect the titer of 1∶128 for a standard serum,which was superior to the result of 1∶64 detected by serum neutralization test.The repeatability test displayed that the co-efficient of variations in intra-and inter-assays were 0.54%to 5.27%and 0.3%to 9.3%,respectively.Comparison of the same sample detection with the commercial kit of ID VET showed that compliance rate was 91.76%.The sensitivity,specificity and compliance of the method reached the application standard.It could be applied for antibody detection and epidemiological investigation for Akabane virus infection in ruminant animals.
作者
苗海生
李乐
廖德芳
寇美玲
朱建波
肖雷
孟锦昕
杨恒
李华春
MIAO Hai-sheng;LI Le;LIAO De-fang;KOU Mei-ling;ZHU Jian-bo;XIAO Lei;MENG Jin-xin;YANG Heng;LI Hua-chun(Yunnan Animal Science and Veterinary Institute,Kunming,Yunnan Tropical and Subtropical Animal Virus Disease Laboratory,Kunming 650224,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2019年第4期371-375,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
云南省技术创新人才培养对象(2018HB076)
国家重点研发计划(2016YFD0500908)
云南省应用基础研究计划(2015FD065)
