摘要
为建立犬瘟热病毒(CDV)的时间分辨免疫荧光分析(TRFIA)方法,本研究将抗CDV的3H7单克隆抗体(MAb)作为包被抗体,Eu3+标记2G4 MAb作为检测抗体,建立了检测CDV抗原的双抗体夹心TRFIA分析法,并初步制备成试剂盒。通过灵敏度、准确度(稀释回收率)、特异性、精密度、稳定性、临床样本检测等实验评价其检测性能。结果显示,该双抗体夹心TRFIA分析法对CDV的检测灵敏度为0.58 ng/mL,线性范围在2.5 ng/mL^640 ng/mL,与荧光值有较强的剂量-反应关系;平均稀释回收率为100.48%,与其它常见病毒无明显交叉反应,特异性较强;批内CV值为5.60%~7.58%,批间CV值为5.67%~7.64%;TRFIA试剂盒可在4℃稳定保存半年,37℃稳定保存7 d。与RT-PCR法同时检测65份疑似CDV样本和15份健康样本,2种检测方法的符合率为100%。本研究建立的CDV TRFIA方法具有灵敏度高、特异性强、准确率好、方便快捷等优点,为大批量CDV临床样品的检测提供了可行技术手段。
To develop a method of time-resolved immunofluorescence analysis(TRFIA)for detecting of canine distemper virus(CDV),meanwhile to preliminary evaluate its capacity of CDV detection,the TRFIA double-antibody sandwich method against CDV was established in this study by using anti-CDV monoclonal antibody clone 3 H7 as the capture antibody and 2 G4 that labeled with the Eu3+as the detection antibody.And then we tested and verified the performances of this method:the sensitivity,accuracy(dilution recovery),specificity,precision,stability,and clinical sample comparison experiments.The results showed that the sensitivity of this double-antibody sandwich TRFIA method was 0.58 ng/mL,and the linear range was between 2.5 ng/mL and640 ng/mL shown strong dose-response relationship with the value of fluorescence signal.The average dilution recovery rate was100.48%without any obvious cross-reaction with other common viruses,which showed high specificity.Besides,the intra-assay precision of this method was between 5.60%and 7.58%,and the inter-assay precision was from 5.67%to 7.64%.In addition,it can be stably stored at 4℃for half a year,and at 37℃for 7 days.Furthermore,65 suspected CDV infection samples and 15 healthy samples were simultaneously detected by RT-PCR and TRFIA,and the coincidence rate was 100%.The CDV TRFIA method established in this study has the advantages of high sensitivity,high specificity,high accuracy and convenience,which is suitable for the detection of large amount CDV clinical samples.
作者
陈翠翠
赖宏锐
梁焕坤
钟树海
黎杰星
李来庆
CHEN Cui-cui;LAI Hong-rui;LIANG Huan-kun;ZHONG Shu-hai;LI Jie-xing;LI Lai-qing(Guangzhou Youdi Biotechnology Co.,Ltd.,Guangzhou 510663,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2020年第3期258-262,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
广州市科技计划项目珠江新星专题(201906010055)
关键词
犬瘟热病毒
双抗体夹心
时间分辨免疫荧光分析
Canine distemper virus
double-antibody sandwich
time-resolved immunofluorescence analysis(TRFIA)
