抗人P185~(erbB_2)单克隆抗体的制备及特性分析

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。

Preparation and identification of monoclonal antibodies against P185~(erbB_2)

Objective To prepare monoclonal antibodies(McAbs)specific to extracellular domain(ECD) of P185 erbB 2 ,and identify high affinity McAbs inhibiting tumor cell proliferation.MethodsBABL/c mice were immunized with SKBR 3cell line highly expressing P185.Hybridoma cell lines were screened by indirect ELISA using ECD of P185 recombinant protein as antigen.ResultsTen hybridoma cell lines were obtained,McAbs recognized only three different epitopes of natural P185.Ig subclass of McAbs were consisted of four IgG1,one IgG3,five IgM.McAbs of four IgG1 inhibit markedly proliferation of SKBR 3 and SKOV 3 cell lines.Immunohistochemistry assay demonstrated McAbs C25 recognized specifically membrane of SKBR 3,SKOV 3 cell lines and breast,ovarian cancer cells,etc.in paraffin sections.ConclusionThese McAbs against P185 may be beneficial to cancer diagnosis,prognosis and cancer therapy.