抗P21蛋白特定位点单克隆抗体的制备及鉴定

PREPARATION AND IDENTIFICATION OF MONOCLONAL ANTIBODIES TO THE DEFINED REGION OF p21 PROTEIN

用相当于ras p21蛋白质氨基酸序列56～64位的合成肽与血蓝蛋白偶联作为抗原,利用B淋巴细胞杂交瘤技术成功地得到了分泌抗P21蛋白的单克隆抗体杂交瘤系SE_(11)及EB_(2411)。其抗体亚型分别为IgG_3及IgG_(2a)。本文着重研究了EB_(2411)单抗与人肿瘤细胞的反应性。免疫组化技术检测结果表明,EB_(2411)单抗与人肝癌_(7402)、乳腺癌_(MCF)、胃癌_(823)、肺腺癌_(A2)及肺鳞癌_(78)等细胞系均呈阳性反应。肺癌、肝癌及结肠癌亦呈现不同程度的阳性反应,而与结肠息肉及肺癌癌旁的正常肺泡的反应为阴性。用放射免疫沉淀-SDS-PAGE技术检测Ha-ras_(12val)转化的细胞HR-1,证明EB_(2411)单抗与p21蛋白反应。

Two hybridoma cell lines SE_(11) and EB_(2411) producing the monoclonal antibodies (McAb) recognizing ras p21 have been generated by lymphocyte hybridization techniques from spleen cells of mice immunized with a synthetic peptide corresponding to amino acids 56～64, a consensus region of three ras gene products. The McAb SE_(11) and EB_(2411) are of the antibody types of IgG_3 and IgG_(2a), respectively. The reactivity fo EB_(2411) with human tumor cells was examined emphatically in this study by immunohistochemical assay. It was shown that EB_(2411) reacted with many human tumor cell lines such as hepatoma_(7402), breast carcinoma_(MCF), stomach carcinoma_(823), lung adenocarcinoma_(A2), and lung squamous carcinoma L_(78), and also with hepatoma, colon carcinoma and lung carcinoma tissues in varying degree. But not with apparently normal pulmonary alveolus adjacent to lung carcinoma and colon polyp tissues. Immunoprecipitation of p12 from normal and c-Ha-ras oncogene transformed NIH 3T3 cells labeled with ^(35)S-methionine and SDS-PAGE showed that p21 in these cells can bedetected by McAb EB_(2411) to a synthetic peptide corresponding to certain sequences of ras gene products. The McAb of p21 obtained may be an useful tool for detecting p21 expression in benign,premalignant and malignant tissues, studying the carcinogenesis mechanism and judging prognosis.