抗人大肠癌单克隆抗体的制备及应用

Preparation and Application of Monoclonal Antibody against Human Colorectal Cancer

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。

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 hands with the relative molecular mass （Mr） 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.