论文部分内容阅读
目的制备可特异识别 P185 erb B2 胞外区的单抗 ,选出亲和力较高并具有抑瘤作用的克隆。方法以高表达 erb B2 的人乳腺癌细胞系 SKBR3免疫 BABL / c小鼠 ,用纯化的重组 DNA表达的 P185胞外区蛋白作为抗原 ,以间接 EL ISA方法筛选阳性克隆。结果获得 10株稳定分泌抗 P185胞外区单抗的杂交瘤细胞株。 4株为 Ig G1、1株为 Ig G3、5株为 Ig M,分别可识别 P185胞外区 3个不同表位 ,且只与天然形式的 P185胞外区特异结合。其中 4株 Ig G1类抗体可明显抑制 SKBR3及 SKOV3细胞增殖。免疫组化实验可使 SKBR3及 SKOV3细胞膜特异性染色 ,且可检出乳腺癌、卵巢癌等石蜡切片标本中 erb B2 高表达的阳性细胞。结论所获单抗可特异识别 P185胞外区抗原 ,具有肿瘤诊断、判断预后及人源化改造后用于治疗的应用潜力。
Objective To prepare monoclonal antibodies that can specifically recognize the extracellular region of P185 erb B2 and select the clones with higher affinity and antitumor activity. Methods BABL / c mice were immunized with SKBR3, a human breast cancer cell line that overexpressed erb B2. The positive clones were screened by indirect ELISA using purified P185 extracellular protein expressed by recombinant DNA as antigen. Results Ten hybridoma cell lines stably secreting monoclonal antibody against P185 extracellular domain were obtained. 4 strains were Ig G1, 1 strain was Ig G3 and 5 strains were Ig M. Three different epitopes were identified in the extracellular region of P185, respectively, and they only specifically bound to the native form of P185 extracellular region. Among them, four strains of Ig G1 antibody significantly inhibited the proliferation of SKBR3 and SKOV3 cells. Immunohistochemical staining could stain the membrane of SKBR3 and SKOV3 cells and detect the high expression of erb B2 in paraffin-embedded specimens of breast cancer and ovarian cancer. Conclusion The McAb can specifically recognize the extracellular region antigen of P185, which has the potential for tumor diagnosis, prognosis and humanized transformation for treatment.