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目的:制备特异性识别天然构象的人表皮生长因子受体-2(HER2)的单克隆抗体(mAb),用以指导Herceptin的临床应用。方法:利用纯化的原核表达的HER2融合蛋白作为抗原免疫BALB/c小鼠,取脾细胞与NS1骨髓瘤细胞按常规方法融合,ELISA法进行杂交瘤的初步筛选。构建HER2酵母展示系统,用酵母cellbase ELISA进行第2次筛选。将筛选到的阳性克隆株进行亚克隆获得稳定分泌细胞株,用免疫组织化学的方法鉴定其与SK-Br-3细胞和293a细胞的反应性。结果:通过ELISA筛选得到168株阳性杂交瘤,用酵母cellbase ELISA最终得到8株阳性杂交瘤,此8株免疫组化检测均与SK-Br-3细胞反应,而与293a细胞无反应。结论:成功制备了8株识别天然构象的HER2的mAb,能够用于免疫组化,为临床上使用Herceptin进行个性化治疗乳腺癌打下了基础。
OBJECTIVE: To prepare a monoclonal antibody (mAb) that specifically recognizes natural conformation of human epidermal growth factor receptor-2 (HER2) to guide the clinical use of Herceptin. Methods: The BALB / c mice were immunized with purified prokaryotic expression HER2 fusion protein. The spleen cells were fused with NS1 myeloma cells by conventional methods. The hybridomas were screened by ELISA. A HER2 yeast display system was constructed and a second screen with yeast cellbase ELISA. The positive clones screened were subcloned to obtain stable secreting cell lines, and their reactivity with SK-Br-3 cells and 293a cells was identified by immunohistochemistry. RESULTS: A total of 168 positive hybridomas were screened by ELISA. Eight positive hybridomas were obtained by yeast cell-based ELISA. All 8 immunohistochemical stains reacted with SK-Br-3 cells and no reaction with 293a cells. CONCLUSION: Eight mAbs that recognize HER2 in their natural conformations were successfully prepared and could be used for immunohistochemistry to lay the foundation for the clinical treatment of breast cancer with Herceptin.