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了解抗人P185erbB2单抗C25的生物学活性,并克隆其可变区基因 ,为研制抗人P185erbB2的人源化抗体奠定基础。方法 :以细胞ELISA、免疫组化等方法分析C25单抗的抗原结合特性 ,用MTT法检测其对乳腺癌细胞SKBR3、及卵巢癌细胞SKOV3 增殖的抑制作用及对化疗药的增敏作用。经RT PCR扩增C25单抗的轻、重链可变区基因 ,克隆后进行核苷酸序列分析。结果 :C25单抗能特异结合人P185erbB2胞外区抗原 ,可显著抑制SKBR3、SKOV3 细胞增殖 ,与顺铂具协同作用。抗体的轻链可变区基因327bp,属于小鼠κ轻链第Ⅲ亚群 ;重链可变区基因366bp属于小鼠IgG1第Ⅲ (A)亚群。结论 :C25单抗可显著抑制人P185erbB2过表达肿瘤细胞的增殖并与顺铂具协同作用 ,所克隆的基因为抗体的可变区基因
Understanding the biological activity of anti-human P185erbB2 monoclonal antibody C25 and cloning its variable region genes laid the foundation for the development of humanized antibodies against human P185erbB2. METHODS: The antigen binding characteristics of C25 monoclonal antibody was analyzed by cell ELISA and immunohistochemistry. The inhibition of proliferation of breast cancer cell SKBR3 and ovarian cancer cell SKOV3 and sensitization of chemotherapeutic drugs were detected by MTT assay. The light and heavy chain variable region genes of the C25 monoclonal antibody were amplified by RT PCR and cloned for nucleotide sequence analysis. RESULTS: The C25 mAb specifically binds to the extracellular domain of human P185erbB2 antigen, and it can significantly inhibit the proliferation of SKBR3 and SKOV3 cells and has a synergistic effect with cisplatin. The light chain variable region gene of the antibody is 327 bp belonging to the mouse kappa light chain III subgroup; the heavy chain variable region gene 366 bp belongs to the mouse IgG1 subgroup III (A) subgroup. Conclusion : C25 mAb significantly inhibits the proliferation of human P185erbB2 overexpressing tumor cells and synergizes with cisplatin. The cloned gene is the variable region gene of the antibody.