抗人大肠癌单克隆抗体的制备及应用

目的制备和纯化抗人大肠癌单克隆抗体(ND-1),分析其在大肠癌诊断中的应用价值。方法常规免疫小鼠制备腹水,腹水经离心和过滤后,应用G蛋白亲和层析法进行纯化。采用SDS-PAGE、间接免疫荧光(IFA)、间接ELISA检测纯化后抗体的纯度、活性、效价和特异性。并用免疫组织化学SP法检测在大肠癌组织中的表达。结果经SDS-PAGE分离可见两条相对分子质量(Mr)分别为55 000和25 000明显的两条蛋白条带。间接ELISA检测抗体效价为1∶1 000 000,ND-1抗体对表达相应抗原的大肠癌细胞株具有特异结合活性。人大肠癌组织切片经ND-1抗体免疫组织化学染色阳性率为82.9%,其中高分化腺癌阳性率为100%。结论应用G蛋白亲和层析法可以很好分离纯化鼠腹水中的IgG1亚型的单克隆抗体,抗体的纯度高,特异性强,生物学活性好,可望成为有效的肿瘤诊断和治疗的手段。

Preparation and Application of Monoclonal Antibody against Human Colorectal Cancer

Objective To prepare and purify a new monoclonal antibody(ND-1) against human colorectal cancer and analyze its application in diagnosis of colorectal cancer.Methods The mouse ascite was prepared by immunizing mice and purified by affinity chromatography using HiTrap protein G column after centrifugation and filtration.The purity,activity,titer and specificity for the purified antibody were determined using SDS-PAGE,indirect immunofluorescence technique and ELISA respectively.The expression of its corresponding antigen in colorectal cancer tissue was tested by immunohistochemical S-P method.Results SDS-PAGE analysis showed that there were two obvious protein bands with the relative molecular mass(M_r) 55 000 and 25 000.ELISA analysis revealed that the titer of the purified antibody was 1∶1 000 000 and had specific binding activity to corresponding antigen-expressing cell lines of colorectal cancer.The positive rate of its corresponding antigen in the tissue of colorectal cancer was 82.9% and that of the well differentiated colorectal cancer was 100%.Conclusion Protein G affinity chromatograpy is effective for the purification of monoclonal antibody ND-1 with high purity,specificity and activity,which is promising means for diagnosis and therapy of tumors.