鸭坦布苏病毒E蛋白Domain Ⅲ单克隆抗体胶体金试纸条的创制及应用  

Development and Application of Colloidal Gold Test Strips Based on Monoclonal Antibodies against Duck Tembusu Virus E Protein Domain Ⅲ

作　　者：阚莹 张淼 卢奇 吕炫 于胜祖 王习强 姜雅倩 王蓓[1] 朱英奇[1] 王晴 王桂军[1] Kan Ying;Zhang Miao;LüQi;Lv Xuan;Yu Shengzu;Wang Xiqiang;Jang Yaqian;Wang Bei;Zhu Yingqi;Wang Qing;Wang Guijun(College of Animal Science and Technology,Anhui Agricultural University,Hefei,Anhui 230036,China)

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《中国动物检疫》2023年第2期131-138,共8页China Animal Health Inspection

基　　金：国家自然科学基金面上项目(32072874);安徽省家禽产业技术体系项目(AHJQCYTX-06-11)。

摘　　要：为建立一种快速诊断鸭坦布苏病毒(DTMUV)的方法,以2株(B9D7B8G10和B9D10C7株)抗DTMUV的单克隆抗体制备DTMUV胶体金免疫层析试纸条。采用经典的柠檬酸钠还原法制备胶体金溶液,利用rProtein G琼脂糖凝胶亲和层析法对两株单克隆抗体腹水进行纯化并鉴定;以纯化的B9D7B8G10株单克隆抗体作为标记抗体,通过调节单克隆抗体浓度与pH确定胶体金探针最佳结合条件;同时,在硝酸纤维素膜(NC膜)检测带包被纯化的B9D10C7株单克隆抗体,质控带包被山羊抗小鼠IgG抗体,建立检测DTMUV胶体金免疫层析试纸条;成功建立试纸条后,对试纸条敏感性、特异性、重复性和稳定性进行检测,并与RT-PCR检测方法进行比较,对200份临床疑似感染样品进行检测。结果显示:制备的胶体金试纸条在15 min内即可完成检测,最低检测稀释度为1:50,且不与鸭瘟病毒、番鸭细小病毒、鹅星状病毒、禽白血病病毒发生交叉反应;应用试纸条和RT-PCR方法分别对200份临床疑似样品进行检测,两者符合率达98%。结果表明,制备的胶体金试纸条敏感性高、特异性强、稳定性好、重复性好,为DTMUV的快速检测提供了一种实用检测技术。In order to develop a rapid diagnostic method for duck Tembusu virus(DTMUV),a colloidal gold immunochromatographic strip was prepared by two strains of monoclonal antibodies(B9D7B8G10 and B9D10C7 strain)against DTMUV.Colloidal gold solution was prepared using classic sodium citrate reduction,and monoclonal antibody ascites of two strains were purified and identified by rProtein G agarose gel affinity chromatography;the optimal binding conditions for colloidal gold probes were confirmed through adjusting the concentration and pH value of the monoclonal antibodies by taking the purified B9D7B8G10 monoclonal antibody as the marker antibody;meanwhile,the purified monoclonal antibodies of B9D10C7 strain with peridium as well as goat anti-mouse IgG antibodies with quality control band peridium were detected on the nitrocellulose membrane(NC membrane)to establish colloidal gold immunochromatographic strips for detecting DTMUV;then the sensitivity,specificity,repeatability and stability of the strips were tested and compared with RT-PCR assay,and 200 clinical samples suspected of DTMUV infection were tested.The results showed that the detection could be completed within 15 min by the prepared strips that were with the minimum detection dilution of 1:50,and failed to crossly react with duck plague virus,muscovy duck parvovirus,goose astrovirus and avian leukemia virus;200 clinical samples were respectively detected by the strips and RT-PCR assay,it was found that the coincidence rate was up to 98%.In conclusion,the prepared strips,as the practical detection technique,could be used to rapidly detect DTMUV with the help of their good sensitivity,specificity,stability and repeatability.

关 键 词：鸭坦布苏病毒 胶体金 单克隆抗体 免疫层析试纸条 

分 类 号：S851.3[农业科学—预防兽医学]