抗登革病毒NS1单抗的制备及检测NS1方法的建立

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/mL NS1。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。

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

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.