草鱼白介素10基因的克隆表达及其双抗体夹心ELISA检测方法的建立

Cloning and expression of Ctenopharyngodon idellus interleukin-10(CiIL-10) gene and establishment of double antibody sandwich ELISA for detection of CiIL-10

为建立检测草鱼白介素10(Ci IL-10)的双抗体夹心ELISA方法,首先克隆Ci IL-10基因、构建与表达原核表达重组质粒p ET-32a-Ci IL-10,并对表达产物进行纯化,以获得重组Ci IL-10(r Ci IL-10)纯化蛋白。然后,以纯化的r Ci IL-10蛋白为免疫原制备兔源及鼠源抗r Ci IL-10的多克隆抗体,并对其进行纯化与酶标记。最后,以纯化的鼠抗r Ci IL-10抗体为捕获抗体,r Ci IL-10纯化蛋白为夹心抗原,HRP标记的兔抗Ci IL-10纯化抗体为检测抗体,通过优化反应条件,建立双抗体夹心ELISA检测方法。结果显示,该方法的优化反应条件包括,捕获抗体的最佳包被浓度为20μg·m L^(-1),检测抗体的最佳稀释度为1∶3 200,最佳封闭条件是使用5%脱脂奶粉,37°C封闭1.5 h,样品反应时间为2h,以OD450值≥0.174作为阳性判定标准。该方法的板内及板间重复性变异系数小于10%,最低检测限量为24.29ng·m L^(-1),与草鱼白介素6、草鱼肿瘤坏死因子、小鼠白介素10、小鼠干扰素和小鼠单核细胞趋化蛋白均无交叉反应。结果表明,建立的双抗体夹心ELISA方法具有较好的特异性、重复性和灵敏度,可用于草鱼白介素10的快速检测。

To establish a double-antibody sandwich ELISA method for the detection of Ctenopharyngodon idellus interleukin 10(CiIL-10). Firstly, CiIL-10 gene was cloned, the prokaryotic expression recombinant plasmid pET-32 a-CiIL-10 was constructed and expressed in the study, and the recombinant CiIL-10(r CiIL-10) protein was purified to obtain the purified protein. Subsequently, rabbit anti-r CiIL-10 polyclonal antibody and mouse anti-r CiIL-10 polyclonal antibody were prepared using the purified r CiIL-10 protein as an immunogen purified and labeled with enzyme. Finally, double-antibody sandwich ELISA(DAS-ELISA) was used, in which the purified mouse anti-r CiIL-10 antibodies, the purified r CiIL-10 protein and the HRP-labeled rabbit anti-CiIL-10 purified antibodies were used as capture antibodies. The sandwich developed the antigen and detected the antibody by optimizing the reaction conditions. The results showed that the optimal reaction conditions of the developed method included coating with the capture antibodies at the concentration of 20 μg·mL^(-1), probing with the HRP-labeled rabbit anti-CiIL-10 antibodies at the proportion of 1∶3 200, sealed with 5% degreasing milk powder at 37 °C for 1.5 hours, 2 hours of reaction time, and judging with OD450 value≥0.174 as a positive criterion. The coefficient of variation of repeatability within and between plates was all less than 10%, in the developed method minimum detection limit was 24.29 ng·mL^(-1). In addition, the established DAS-ELISA could detect CiIL-10 specifically, no cross-reaction with other cytokines including grass carp interleukin-6, grass carp tumor necrosis factor, mouse interleukin-10, mouse interferon, or mouse monocyte chemoattractant protein. Our results indicated that the established double-antibody sandwich ELISA in this study possessed a better specificity, reproducibility and sensitivity, which might be used for the rapid detection of CiIL-10.