hMIC1抗体的研制 鉴定及胰腺癌患者血清中MC1升高的临床价值.docx

北京协和医学院硕士学位论文摘 北京协和医学院硕士学位论文 摘 要 第一部分hMIC-1抗体的研制及鉴定 目的：制备人巨噬细胞抑制因子一1(hMIC-1)的单克隆抗体与多克隆抗体，为建立MIC-1 免疫学检测方法和功能研究奠定物质基础。 方法：以酵母表达的hMIC一1重组蛋白作为免疫原免疫BALB／cd、鼠，通过传统杂交瘤技术制 备抗hMIC—l单克隆抗体，采用间接ELISA和Western blot等方法鉴定抗体的亚类、特异 性等特性。采用免疫组织化学等常规检测方法进一步检验抗体的特异性及可能的用途。此 外，将hMIC-1重组抗原经多次多点免疫新西兰大耳白兔，制备得到兔抗hMIC-1抗血清，纯 化后鉴定抗体的特异性，并初步探索建立ELISA检测方法的可行性。 结果：获得l株可稳定分泌抗hMIC—l单克隆抗体的杂交瘤细胞株，命名为2G7。Western blot鉴定表明2G7单抗能够特异识别重组hMIC-1及人结肠组织表达的还原型与非还原型 MIC-1，与酵母粗提取物无交叉反应。 应用2G7抗体免疫组化检测MIC-1在结肠癌、前列腺 癌表达情况，结果表明MIC—1分布于细胞浆，呈强阳性着色，与商售MIC-1单抗相一致。应 用2G7抗体的免疫组化片背景更低，显示出2G7抗体具有较好的特异性。兔抗重组hMIC一1多 克隆抗体能够识别天然hMIC一1，血清抗体效价为105，但不能与2G7单抗配对检测hMIC—l。 结论：以酵母表达的重组hMIC-1作为免疫原能够用于制备抗hMIC-1单克隆抗体杂交瘤。本 研究获得了1株针对hMIC-1的单克隆抗体瘤株，产生的抗体能应用于hMIC一1 Western blot 检测。 关键词： MIC-1单克隆抗体杂交瘤多克隆抗体 2 北京协和医学院硕十学位论文Preparation 北京协和医学院硕十学位论文 Preparation and identification of antibody against hMIC-1 Objective：To make and identify the antibody against hMIC-1 for application in immunoassay and study of gene functions． Methods：BALB／c mice were immunized with the recombinant hMIC一1 protein expressed in Pichia Pastoris，and monoclonal antibody(McAb)against hMIC-1 was produced by the classical hybridoma technique．Polyclonal antibody against hMIC一1 was obtained from New Zealand White rabbits immunized by multi-site,s and multi．time periods．These anti．hMIC．1 antibodies were characterized by EtaSA,Western blot and immunohistochemical staining．Furthermore，a enzyme-linked immtmosorbent assay for the quantification of serum hMIC·1 was developed． Results：A hybridoma line Was successfully established to secrete the McAb against hMIC· 1,which named 2G7．By Western blot，it Was found that the McAb 2(37 recognized the reducing and noreducing hMIC-1 in colon cancers，and had no cross-reaction with Pichia Pastoris crude extract．The characterized McAb Was applied for immunohistochemical staining in colon啪oe璐 and prostate cancer，and displayed strong specificity and moderate affinity compared with the commercial McAb．The results showed that MIC-1 staining distrib