单抗介导的银加强金标免疫技术检测传染性支气管炎病毒  

Detection on antigen of infectious bronchitis virus in chicken by silver-enhanced colloidal gold assay

作　　者：王新卫[1,2] 何宏轩[1] 王泽霖[2] 吴艳云[1] 王承民[1] 

机构地区：[1]中国科学院动物所动物生态与保护生物学院重点实验室国家野生动物疫病研究中心,北京100101 [2]河南农业大学牧医工程学院,河南郑州450002

出　　处：《中国兽医学报》2009年第4期394-398,共5页Chinese Journal of Veterinary Science

基　　金：国家“863”计划资助项目(2007AA100606);WCS项目(ECBP/06/085)

摘　　要：利用胶体金标记纯化的羊抗鼠IgG，建立了一种银加强金标免疫技术（SECGA）检测IB抗原。检测抗原时先将抗原点样于硝酸纤维素膜（NCM）中央，封闭后浸于l：40IBV单抗中作用10min，洗涤后再浸于1：4金标羊抗鼠IgG液中作用1h，银染10min后观察，在膜上出现灰黑斑点的为阳性结果，反之，不出现斑点为阴性结果。SECGA对IB抗原最低检测量为0．703ng／点（2μL），对含毒尿囊液的最低检测量为10^2.9EID50，检测IBV M41在鸡胚中繁殖时以36-54h含毒量最高；检测SPF鸡气管粘液，至少于感染后3d内检出病毒。结果表明，SECGA检测抗原可用于IBV早期诊断，具有群特异性、快速、敏感、简便，不需特殊设备、结果易判定等优点，尤适于基层实验室或现场应用。The purified goat anti-rat IgG was labeled with colloidal gold particals of 10 nm in diameter. A silver-enhanced colloidal gold assay （SECGA）was developed for detection of IBV in chicken. When IBV antigen was detected,nitrocellulose membrane （NCM） with IBV was blocked and reacted in solution of anti-IBV McAb diluted with 1 ： 40 for 10 minutes. After being washed, NCM was immersed in the solution of goat anti-rat IgG labeled with colloidal gold diluted with 1 ： 4 for one hour. Then NCM with sample was stained 10 minutes by silver. The sample with clear and obvious black dot was judged as positive reaction for IBV, otherwise, negative result. The identification-limit of purified IBV antigen and allantoic fluid of IBV were 0. 703 ng with each dot （0. 351 5 mg/L）, 102.9 EID50 respectively. IBV titer in inoculated eggs was highest from 36 to 54 hours post inoculation （p. i. ）. IBV could be detected in trachea of specific pathogen free （SPF） chicks in 3 days p. i. by using SECGA at least. This indicated that SECGA could be applied to diagnose IBV early, which was group-specific, rapid, sensitive, simple. What＇ s more, it needed no particular instruments and the result was determined easily. SECGA could be performed especially in clinical diagnosis and primary laboratory.

关 键 词：传染性支气管炎病毒 单克隆抗体 胶体金 银加强胶体金检测技术 

分 类 号：S852.65[农业科学—基础兽医学]