论文部分内容阅读
目的 :制备鼠抗人白细胞介素 15(hIL 15)单克隆抗体 (mAb) ,并鉴定其特性。方法 :自重组人白细胞介素 15(rhIL 15)基因工程菌中 ,提取融合蛋白GST IL 15,以 12 0g/LSDS PAGE分离鉴定 ,切取含有目的条带的凝胶 ,免疫BALB/c小鼠。取免疫小鼠的脾细胞与Sp2 / 0骨髓瘤细胞常规融合 ,依次进行HAT选择培养 ,间接ELISA法筛选抗体阳性的杂交瘤细胞及克隆化。对杂交瘤细胞株的稳定性及其分泌的mAb的特性进行鉴定。另外 ,以rhIL 15包涵体蛋白 (rhIL 15IBP)免疫新西兰白兔 ,制备抗hIL 15的多克隆抗体 (多抗 )。用抗hIL 15的mAb与多抗建立双抗体夹心间接ELISA。结果 :获得 1株可稳定分泌特异性抗hIL 15mAb的杂交瘤细胞。建立了双抗体夹心间接ELISA ,检测rhIL 15蛋白的敏感性达 10 μg/L。结论 :成功地制备抗hIL 15mAb ,并建立了一种可用于hIL 15检测的双抗体夹心间接ELISA。
Objective: To prepare and characterize murine anti-human interleukin 15 (mAb) monoclonal antibody (mAb). Methods: Recombinant human interleukin - 15 (rhIL - 15) was used to isolate GST IL 15 fusion protein. The recombinant protein was isolated and identified by 12 0 g / L SDS PAGE. The gel containing the target band was excised and BALB / c mice were immunized. The spleen cells of the immunized mice were routinely fused with Sp2 / 0 myeloma cells, followed by HAT selection culture, and the antibody-positive hybridoma cells were screened by indirect ELISA and cloned. The stability of the hybridoma cell line and the characteristics of the secreted mAb were identified. In addition, New Zealand white rabbits were immunized with rhIL 15 inclusion body protein (rhIL 15IBP) to prepare a polyclonal antibody (polyclonal antibody) to hIL 15. A double antibody sandwich indirect ELISA was established with mAb against hIL15 and polyclonal antibodies. Results: One hybridoma cell line which can stably secrete specific anti-hIL 15 mAb was obtained. A dual-antibody sandwich indirect ELISA was developed to detect the sensitivity of rhIL 15 protein to 10 μg / L. Conclusion: The anti-hIL 15 mAb was successfully prepared and a double-antibody sandwich ELISA was developed for the detection of hIL 15.