Wang Ping, Wang Run-tian, Wang Li, Wang Cong-yin, Tong Hui, Li Quan-hai, Yang Li-juan
Department of Immunology, Institute of Basic Medical Sciences, Hebei Medical University, Shijiazhuang 050017, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2004 Mar;20(2):183-5.
To prepare monoclonal antibody (mAb) against human interleukin-15 (hIL-15) and identify its characterization.
The GST-IL-15 was extracted from the gene-engineering bacteria E. coli and identified by SDS-PAGE. The gel strip containing GST-IL-15 was cut off to immunize BALB/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 hybridoma cells secreting specific antibody were detected by ELISA and cloned by limiting dilution. The stability of the obtained hybridoma cells and the specificity of anti-hIL-15 mAb the hybridoma cells secreted were identified. In addition, the New Zealand rabbits were immunized with the rhIL-15 inclusion body protein (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 coating and sandwich antibodies, respectively, to detect hIL-15.
One hybridoma cell line which could stably secrete specific mAb was obtained. A sandwich ELISA for detecting rhIL-15 protein was established and its sensitivity was as low as 10 microg/L.
The anti-hIL-15 mAb was prepared successfully. A sandwich ELISA for the detection of hIL-15 was established.
制备抗人白细胞介素-15（hIL-15）单克隆抗体（mAb）并鉴定其特性。
从基因工程菌大肠杆菌中提取谷胱甘肽S-转移酶-白细胞介素-15（GST-IL-15），并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行鉴定。切下含有GST-IL-15的凝胶条带用于免疫BALB/c小鼠。采用常规方法将免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合，并在次黄嘌呤氨基蝶呤胸腺嘧啶核苷（HAT）培养基中筛选杂交瘤细胞。通过酶联免疫吸附测定（ELISA）检测分泌特异性抗体的杂交瘤细胞，并通过有限稀释法进行克隆。鉴定所获得杂交瘤细胞的稳定性以及该杂交瘤细胞分泌的抗hIL-15单克隆抗体的特异性。此外，用重组人白细胞介素-15包涵体蛋白（rhIL-15IBP）免疫新西兰兔，制备抗hIL-15多克隆抗体（pAb）。分别以抗白细胞介素-15单克隆抗体和多克隆抗体作为包被抗体和夹心抗体，建立夹心ELISA法检测hIL-15。
获得了一株能够稳定分泌特异性单克隆抗体的杂交瘤细胞系。建立了用于检测重组人白细胞介素-15蛋白的夹心ELISA法，其灵敏度低至10微克/升。
成功制备了抗hIL-15单克隆抗体。建立了用于检测hIL-15的夹心ELISA法。
Zotero 插件