PEDV IgG抗体时间分辨荧光免疫层析(TRFIA)检测方法的建立  

Establishment of time-resolved fluorescence immunochromatographic assay for detection of PEDV IgG antibody

作　　者：朱玲 程育芳 曹华 陈淑华[2] 孟宪荣[2] 何启盖[2] ZHU Ling;CHENG Yu-fang;CAO Hua;CHEN Shu-hua;MENG Xian-rong;HE Qi-gai(State Key Laboratory of Agricultural Microbiology,Huazhong Agricutural XJniuersity Wuhan 430070 iChina;College of Animal Science and Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070 China)

机构地区：[1]华中农业大学农业微生物学国家重点实验室,湖北武汉430070 [2]华中农业大学动物医学院,湖北武汉430070

出　　处：《中国兽医学报》2020年第11期2085-2089,共5页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2016YFD0500702)

摘　　要：为了建立一种快速定量检测血清中猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)IgG抗体时间分辨荧光免疫层析(TRFIA)的检测方法,本研究采用竞争法和荧光免疫层析技术,以羧基荧光微球与PEDV S蛋白偶联,以抗IgG FC片段单抗和PEDV S1单抗作为试纸条检测线(T线)和质控线(C线),制备PEDV IgG抗体检测荧光定量免疫层析试纸条。通过对蛋白与荧光微球偶联方法、包被抗体量等对本研究工艺进行优化,并通过线性范围、最低检出限、精密性等性能指标以及与临床样品检测结果比对,对PEDV IgG抗体时间分辨荧光层析检测方法进行评价。结果显示,本研究所制备的PEDV IgG抗体检测试纸条线性范围为25~2 500 g/L,低检出限为25 g/L,批内变异系数与批间变异系数均小于15%,平均添加回收率为100.88%~102.70%,检测时间为15 min,试纸条与5种其他猪病血清抗体无交叉反应。临床样品检测发现,其与实验室间接免疫荧光检测结果符合率为95.6%。综上,本研究所研制的PEDV IgG抗体检测时间分辨荧光免疫层析试纸条灵敏度较高、特异性较高、准确度高、操作简便、快速,可用于临床PEDV IgG抗体的检测。In order to establish a rapid quantitative assay for the detection of PEDV IgG antibody in serum by time-resolved fluorescence immunochromatography assay(TRFIA),this study used competition method and fluorescence immunochromatography technique,carboxy fluorescent microspheres were coupled with PEDV S protein,anti-IgG FC fragment monoclonal antibody and PEDV S1 monoclonal antibody were used as test strip detection line(T-line) and control line(C-line) to preparae PEDV IgG antibody fluorescence quantitative immunochromatographic test strip.Optimized the research process by adjusting the amount of coupling the protein to the fluorescent microspheres,coating the antibody amount,etc.Accoording to the linear range,the lowest detection limit,the precision and the performance of the clinical sample,was the time-resolved fluorescence immunochromatographic assay of PEDV IgG antibody was evaluated.The results showed that the detection range of PEDV IgG antibody test strip was 25-2 500 g/L,the lowest detection limit was 25 g/L,and the CV in intra-assay and inter-assay was less than 15%.The average recovery was 100.88% to 102.70%,the detection time was 15 min,and there was no cross-react to the serum antibodies of five other swine diseases.Clinical sample detection found that the coincidence rate with laboratory indirect immunofluorescence assay results was 95.6%.In summary,the timeresolved fluorescence immunochromatographic detection method of PEDV IgG antibody developed in this study has higher sensitivity,higher specificity,high degree of accuracy,easy and fast to operation,can be used for the detection of clinical PEDV IgG antibodies.

关 键 词：猪流行性腹泻(PED) IGG抗体 时间分辨荧光免疫层析(TRFIA) 定量检测 快检 

分 类 号：S852.65[农业科学—基础兽医学]