抗登革病毒NS1单抗的制备及检测NS1方法的建立  

Preparation of the Monoclonal Antibodies against Dengue Virus NS1 Protein and Establishment of a Capture ELISA to Detect Dengue Virus NS1

作　　者：郝永华[1] 王芹[1] 关武祥[1] 修梅红[1] 戴晓霞[1] 郑峰[1] 李伟红[1] 刘峰[1] 刘琴芝[1] 李川[1] 张全福[1] 梁米芳[1] 李德新[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,北京100052

出　　处：《病毒学报》2006年第4期292-296,共5页Chinese Journal of Virology

摘　　要：制备抗登革病毒NS1蛋白单克隆抗体，建立检测NS1的ELISA方法。表达1～4型登革病毒NS1蛋白，将1型NS1蛋白纯化后免疫BALB／c小鼠，通过杂交瘤技术制备单克隆抗体。经ELISA、Western blotting、间接免疫荧光筛选和鉴定单克隆抗体，进行纯化和HRP标记。通过鉴定每两株单抗之间是否存在竞争作用，选择非竞争单抗组合并建立NS1捕获法ELISA。结果获得7株高滴度抗NS1单抗，捕获法ELISA可以检出10ng／mLNS1。原核表达登革病毒NS1蛋白制备的单抗可以和天然病毒抗原反应，NS1捕获法ELISA可以用于登革病毒感染检测。For preparing the monoclonal antibodies against Dengue virus NS1 protein and developing an ELISA method to detect Dengue virus NS1, NS1 genes of serotypes 1 -4 of Dengue virus were amplified by RT-PCR. PCR products were cloned into pET30a vectors. The NS1 proteins of Dengue virus serotypes 1 - 4 were expressed and purified. BALB/c mice were immunized with recombinant NS1 of Dengue virus serotype 1. The spleen cells of the immunized mice were fused with SP2/0 cells. The antibody-secreting hybridomas were tested by ELISA, Western blotting and indirect immunofluorescence assay. Then the secreted monoclonal antibodies were purified by protein G and labeled with HRP. Every two antibodies were combined and tested if they competed. The non-competed antibodies were combined for optimizing the NS1 capture ELISA. Seven monoclonal antibodies of high titers were obtained. The sensitivity of the NS1 capture ELISA was up to 10 ng/mL. The NS1 capture ELISA can be used for differentiating between Dengue virus and Japanese encephalitis virus infections.

关 键 词：登革病毒 NS1蛋白 单克隆抗体 

分 类 号：R373.22[医药卫生—病原生物学]