亲和层析法纯化抗大肠肿瘤相关抗原的单克隆抗体

Purification of monoclonal antibody against colorectal tumor-associated antigen by protein G affinity chromatography

目的制备和纯化鼠源性抗人大肠肿瘤单克隆抗体(单抗),分析其相应抗原在大肠肿瘤细胞中的表达。方法常规方法免疫BALB/c小鼠制备腹水,应用G蛋白亲和层析柱对小鼠腹水样品进行分离纯化。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。用免疫细胞化学、流式细胞术和免疫组织化学技术S-P法检测其相应抗原在大肠肿瘤细胞株和组织中的表达。结果还原性SDS-PAGE显示所获抗体为单一组分。应用间接ELISA检测抗体效价为1∶106,纯化后的抗体对大肠肿瘤细胞株具有特异结合活性,并在高分化大肠腺癌组织中表现出更高选择性(P<0.01)。结论G蛋白亲和层析法操作简便,所得单抗的纯度高,特异性强,生物学活性好。对临床上早期诊断大肠肿瘤将具有较大的意义。

Objective To prepare and purify a new monoclonal antibody（ND-1） against human colorectal carcinoma and analyze its corresponding antigen expression in colorectal carcinoma cells. Methods BALB/c mice were immunized by routine technique to prepare ascites , and the ascites samples were purified using protein G affinity chromatography column. The purity, activity, titer and specificity of the purified antibody were detected by SDS-PAGE, indirect irnmunofluorescence technique and indirect ELISA, respectively . Then the expression of its corresponding antigen in colorectal carcinoma cell lines and tissues was tested through immunocytochemical staining, flow cytometry and immunohistochemistry S-P method. Results Reductive SDS-PAGE showed that purified antibody was a single component. The titers of the purified antibody were 1 ： 10^6 by ELISA. The ND-1 IgG had specific binding activity to colorectal carcinoma cell lines and exhibited higher selectivity in high differentiated colorectal adenocarcinoma. Conclusion Protein G affinity chromatography method is simple and suitable for purification of monoclonal antibody. The antibody obtained is of high purity, specificity and biological activity, thus it is very important for early diagnosis of colorectal carcinoma.