抗肿瘤坏死因子相关凋亡诱导配体细胞外段单克隆抗体的制备

Preparation of Monoclonal Antibody against Extracellular Domain of TNF-related Apoptosis-induced Ligand

目的制备抗肿瘤坏死因子相关凋亡诱导配体细胞外段(sTRAIL)的单克隆抗体。方法用重组sTRAIL免疫BALB/c小鼠,取脾细胞与SP2/0细胞融合,捕获ELISA筛选阳性克隆,制备腹水抗体。间接ELISA测定单抗效价,并进行单抗亚类、特异性鉴定及杂交瘤细胞染色体和单抗识别位点分析。将杂交瘤细胞体外连续培养3个月及冻存6个月后,检测细胞培养上清的效价。结果3株杂交瘤细胞分泌的单抗均为IgG1型,腹水单抗效价均高于10-6,杂交瘤细胞染色体数介于94～98条之间,均识别sTRAIL分子上线性表位。细胞体外连续培养3个月及冻存6个月后的上清效价保持稳定。结论已成功制备了3株可稳定分泌抗sTRAIL单克隆抗体的杂交瘤细胞,为TRAIL的定性定量检测及组织表达分析奠定了基础。

Objective To prepare the monoclonal antibody（McAb）against amino acids 114-281 at extracellular domain of TNF-related apoptosis-induced ligand （TRAIL）, i.e. sTRAIL. Methods BALB / c mice were immunized with recombinant sTRAIL, and their splenic cells were fused with SP2 / 0 cells, and positive clones were screened by capture ELISA to prepare McAb in ascites. The prepared McAb was determined for titer by indirect ELISA and analyzed for subclass and specificity. Meanwhile, the chromosome of hybridoma cells and recognition site of McAb were analyzed. The culture supernatants of hybridoma cells 3 months after culture in vitro and 6 months after storage in frozen were determined for McAb titers respectively. Results All the McAbs secreted by 3 hybridoma cell strains were IgG1, and their titers in ascites were higher than 10-6. The number of chromosome in hybridoma cells was 94～98. All the McAbs recognized the linear epitopes on sTRAIL molecules. The McAb titers in culture supernatants of hybridoma cells showed no significant change after culture in vitro for 3 months or storage in frozen for 6 months. Conclusion Three hybridoma cells stably secreting McAbs against sTRAIL were successfully established, and McAbs were prepared, which laid a foundation of qualitative and quantitative assays as well as expression of TRAIL.