抗人白细胞介素15单克隆抗体的制备及特性鉴定

Preparation and characterization of anti-human IL-15 monoclonal antibody

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。

AIM: To prepare monoclonal antibody (mAb) against human interleukin-15 (hIL-15) and identify its characterization. METHODS: The GST-I L-15 was extracted from the gene-engineering bacteria E.coli and identifie d by SDS-PAGE. The gel strip containing GST-IL-15 was cut off to immunize BAL B/c mice. The splenocytes of immunized mice were fused with Sp2/0 myeloma cells by a routine method and the hybridomas were selected in HAT medium. The hybridom a cells secreting specific antibody were detected by ELISA and cloned by limitin g dilution. The stability of the obtained hybridoma cells and the specificity o f anti-hIL-15 mAb the hybridoma cells secreted were identified. In addition, t he New Zealand rabbits were immunized with the rhIL-15 inclusion body pr otein (rhIL-15IBP) to prepare the polyclonal antibody (pAb) against hIL- 15. A sandwich ELISA was established with the anti-IL-15 mAb and pAb as coatin g and sandwich antibodies, respectively, to detect hIL-15. RESULTS: One hybridoma cell line which could stably secrete specific mAb was obtaine d. A sandwich ELISA for detecting rhIL-15 protein was established and i ts sensitivity was as low as 10 μg/L. CONCLUSION: The an ti-hIL-15 mAb was prepared successfully. A sandwich ELISA for the detection of hIL-15 was established.