银加强金标免疫技术在鸡传染性支气管炎病毒检测中的应用  

Application of Silver-enhanced Colloidal Gold Assay on Detection of Infectious Bronchitis Virus in Chicken

作　　者：王泽霖[1] 王新卫[1] 刘守川[1] 陈少渠[1] 杨卫华 

机构地区：[1]河南农业大学禽病研究所,河南郑州450002 [2]郑州市原种场,河南郑州450007

出　　处：《河南农业大学学报》2005年第2期201-205,共5页Journal of Henan Agricultural University

基　　金：内蒙古金宇集团股份公司科研基金资助(04544)

摘　　要：采用胶体金标记纯化的羊抗鼠IgG,建立了一种对鸡传染性支气管炎病毒(IBV)进行检测的银加强金标免疫技术(SECGA).先将抗原点样于硝酸纤维素膜(NCM)中央,封闭后浸于40×稀释的IBV单抗中作用10min,洗涤后再浸于体积比为1:4稀释的金标羊抗鼠IgG溶液中作用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抗原最低测量值为0.703ng·点-1(约2μL),对含毒尿囊液的最低检测为102..9EID50.结果表明,IB VM41毒株在鸡胚中繁殖时以36～54h检测含毒量最高;SPF鸡感染3d后可从气管粘液中检出病毒.SECGA检测抗原可用于IBV早期检测,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 10nm in diameter.A Silver-Enhanced Colliodal Glod assay was developed for detection of IBV in chicken.When IBV was detected,nitrocellulose membrane(NCM) with IBV to be detected 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 for 10 minutes by silver.The sample with clear and obvious black dot was judged as positive reaction for IBV,otherwise,negative result.The detection limit of purified IBV antigen and allantioic fluid of IBV were 0.703ng with each dot(0.351 5 μg·mL^(-1)),and 10^(2.9) EID(50) respectively.The results show IBV titre in inoculated eggs was highest from 36 to 54 hours post inoculation(p.i.).IBV could be detected in 3 days p.i. in trachea of specific pathogen free(SPF) chicks by SECGA at least.SECGA could be applied to (diagnose) IBV at the early stage and used for antibody surveillance of IB.It was group-specific,rapid,sensitive,and simple.What's more,it does not need particular instruments and the result was determined easily and so on.SECGA could beperformed especially in clinical diagnosis and primary laboratory.

关 键 词：免疫技术 病毒检测 鸡传染性支气管炎病毒 硝酸纤维素膜 胶体金标记 SPF鸡 早期检测 检测抗原 现场应用 IgG IBV 体积比 测量值 B抗原 尿囊液 含毒量 特异性 实验室 稀释 斑点 单抗 鸡胚 毒株 羊 

分 类 号：S858.31[农业科学—临床兽医学] TP309[农业科学—兽医学]