鸭新城疫病毒单克隆抗体的制备及双抗体夹心ELISA方法的初步建立  

Preparation of Monoclonal Antibody Against Duck Newcastle Disease Virus and Establishment of a Sandwich Enzyme-Linked Immunosorbent Assay

作　　者：孙敏华[1] 董嘉文[1] 吕殿红[1] 周秀蓉[1] 李林林[1] 胡奇林[1] 

机构地区：[1]广东省农业科学院兽医研究所广东省公共卫生公共实验室,广东广州510640

出　　处：《动物医学进展》2012年第12期54-58,共5页Progress In Veterinary Medicine

基　　金：广东省农业攻关项目(2007A020030005-12);广东省兽医公共卫生公共实验室开放基金项目(GSKJ090201)

摘　　要：为了对目前流行的鸭新城疫进行快速诊断,应用蔗糖密度梯度离心纯化后的鸭新城疫病毒免疫6周龄雌性Balb/c小鼠,取免疫后小鼠脾细胞与骨髓瘤细胞SP2/0融合。采用ELISA方法筛选阳性细胞克隆,经3次亚克隆后获得一株能稳定分泌鸭新城疫病毒特异性单克隆抗体的细胞株2C1。以PEG纯化后的2C1腹水为捕获抗体,辛酸-硫酸铵纯化的兔抗鸭新城疫病毒多克隆抗体为第二抗体,HRP标记的羊抗兔抗体作为酶标抗体,建立了检测鸭新城疫病毒抗原的双抗体夹心ELISA方法。结果表明,当2C1包被量为4μg/孔,鸭新城疫病毒1∶10稀释,兔抗鸭新城疫病毒多抗作用量为0.1μg/孔,HRP标记的羊抗兔二抗1∶10 000倍稀释,每次作用时间为1h时,P/N比值最高,且阳性OD值最接近1.0。试验表明,该方法特异性好,不与IBV、ARV、GPV、DPV、DHV尿囊液及H5、H9标准阳性抗原发生反应;可以检测出0.2μg纯化病毒,比传统的HA试验的敏感性至少高64倍;批间和批内变异系数均小于10%。本研究建立的双抗体夹心ELISA方法为鸭新城疫病毒的快速诊断奠定了基础。For rapid testing duck newcastle disease virus （NDV）, one hybridoma cell strain was developed by confusion SP2/0 cells and spleen cells of immunized Balb/c female mouse with duck NDV, which was purified by sucrose density gradient eentrifugation. A positive hybridoma cell strain 2C1 underwent three times subcloning, was identified. Subsequently, the sandwich ELISA was developed as follows： the ascites fluid of 2C1 purified by PEG （polyethylene glycol） was used as coating antibody, and the polyclonal antibody against duck NDV precipitated with eaprylic acid and ammonium sulfate was used as the secondary antibody, and the peroxidase-conjugated goat anti-rabbit IgG（H＋L） was performed as enzyme labeled antibody. The results showed that optimal working conditions, P/N value maximized, were the case that 2C1 coated with a dosage of 4 microgram per well （tag/well）, duck NDV underwent a ten fold dilution, polyclonal antibody gave a dosage of 0.1 μg/well, and enzyme labeled antibody diluted to 1 ＂ 10 000, each of them incubated one hour. The high specificity made the assay reacted with none of these viruses, namely infectious bronchitis virus, avian reovirus, goose parvovirus, duck plague virus, duck hepatitis virus and standard antigen of HS, H9 avian influenza virus. The sensitivity of the sandwich ELISA was 64-fold higher than HA test, and the detection threshold was as low as 0.2 tag purified duck NDV. The coefficient of variability was lower than 10G. In conclusion, this work made the rapid test for duck NDV possible.

关 键 词：鸭新城疫毒 单克隆抗体 夹心ELISA 

分 类 号：S852.659.5[农业科学—基础兽医学]