新城疫病毒流式微球免疫检测新方法  

A Novel Immunoassay of Detecting Newcastle Disease Virus by Cytometry with Flowing Microsphere as Protein Carrier

作　　者：杨丽丽[1] 王振国[2] 王明泰[2] 牟峻[2] 邹明强[1] 李锦丰[1] 王楠[1] 钱爱东[3] 

机构地区：[1]中国检验检疫科学研究院,北京100025 [2]吉林出入境检验检疫局,长春130062 [3]吉林农业大学动物科技学院,长春130118

出　　处：《分析化学》2008年第1期29-33,共5页Chinese Journal of Analytical Chemistry

基　　金：科技部科研院所社会公益研究专项(No.2005DIA2J128);科技部防控禽流感应急科技专项(No.2004BA519A56);国家质检总局科技攻关项目(No.2003IK057、2004IK033、2005IK0073)资助

摘　　要：本研究基于新城疫病毒多克隆抗体的制备及优化,以聚苯乙烯微球作为蛋白质载体建立免疫检测体系,建立了快速检测新城疫病毒的流式细胞术新方法。以藻红蛋白荧光染料对新城疫多克隆抗体荧光标记,取1μL荧光微球与100μL新配制的单克隆抗体溶液反应,依次加入一定量待测抗原和藻红蛋白标记的多克隆抗体,漩涡振荡10s,室温振荡反应充分,形成双抗体夹心复合物,用流式细胞仪进行检测。采用ELISA法对免疫试剂进行了匹配性筛选实验,优选了试剂用量,并与传统ELISA方法进行了对比分析。实验结果表明,单克隆抗体350mg/L、多克隆抗体300mg/L时可获得最佳分析效果,与传统的ELISA方法呈现出良好的相关性,不与鸡传染性支气管炎病毒、鸡痘病毒、鸡马立克氏病病毒等发生交叉反应。A novel immunoassay for detecting the Newcastle disease virus（NDV） by cytometry with flowing fluorescence-coded microsphere as protein cattier was studied after the polyclonal antibody （PcAb） was prepared and purified. In the test, PcAb of NDV was labeled with R-phycoerythrin（R-PE）. Sampled 1 μL of fluorescent microsphere to 100 μL of the monoclonal antibody solution newly prepared, added a certain amount of antigen and R-PE PcAb, vortexed immediately for 10 s. The mixture was thoroughly incubated on a rocker at room temperature, and a sandwich of double antibodies was formed, then the aliquot was detected by flow cytometry. The specificity, stability and sensitivity of the method were respectively examined. As an optimization result, monoclonal antibody （McAb） concentration of 350 mg/L and PcAb concentration of 300 mg/L were selected. The results were compared with traditional method and it was found that the established method was well relative with conventional ELISA, without cross-reaction with infectious bronchitis virus （IBV）, infectious bursal disease virus （IBDV） , egg drop syndrome＇76 （EDS76） and marek＇s disease virus （MDV）.

关 键 词：新城疫病毒 流式细胞术 微球 免疫检测 

分 类 号：R446.6[医药卫生—诊断学]