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目的:制备针对EGFRvⅢ的单克隆抗体,以期待其对消化道肿瘤的靶向治疗。方法:通过人工合成EGFRvⅢ与KLH连接,免疫Balb/c小鼠,采用杂交瘤技术制备鼠源性单克隆抗体细胞株,经腹水生产抗体,并对抗体进行纯化和相关性质鉴定。结果:合成EGFRvⅢ的基因缺失融合区的相应14肽,短肽与蛋白载体KLH连接作为抗原,免疫Balb/c小鼠。制备针对表达EGFRvⅢ肿瘤而对正常组织无破坏作用的的单克隆抗体。通过常规免疫程序获得高效价(1∶128 000)抗血清的免疫小鼠后,进行细胞融合,制备杂交瘤单克隆抗体细胞。应用ELISA检测的方法筛选鉴定能分泌EGFRvⅢ抗体的杂交瘤细胞株,经亚克隆获得5株鼠源性成系抗体阳性细胞株。阳性细胞株经扩大培养,注射于小鼠腹腔以大量制备抗体。抗体型别鉴定为IgG2a,并对抗体效价及其相对亲合力进行测定。腹水效价达1∶128 000,亲和力最高达9.8×10-9 mol/L。用表达的正常EGFR配体结合区蛋白检测自制抗体的特异性,结果显示抗体不与正常EGFR配体结合区蛋白结合。结论:成功制备了抗鼠EGFRvⅢ单克隆抗体,为后续抗肿瘤的靶向治疗奠定基础。
OBJECTIVE: To prepare a monoclonal antibody against EGFRvIII in the hope of targeting the treatment of gastrointestinal tumors. Methods: Balb / c mice were immunized with synthetic EGFRv Ⅲ and KLH, and murine monoclonal antibody cell lines were prepared by hybridoma technique. Antibodies were produced by ascites fluid and the antibodies were purified and identified. Results: The corresponding 14-mer of the fusion gene of EGFRvⅢ gene was deleted and the short peptide was linked with the KLH protein carrier as an antigen to immunize Balb / c mice. Monoclonal antibodies against the expression of EGFRvIII tumor without damage to normal tissues were prepared. After obtaining high titer (1: 128,000) antiserum-immunized mice by conventional immunization procedures, cell hybridization was performed to prepare hybridoma monoclonal antibody cells. The hybridoma cell lines secreting EGFRvIII antibody were screened by ELISA. Five murine hybrid strains were obtained by subcloning. Positive cell lines were expanded and injected into the abdominal cavity of mice to prepare antibody in large quantities. The antibody type was identified as IgG2a and the antibody titer and its relative avidity were determined. Ascites titer up to 1: 12800, affinity up to 9.8 × 10-9 mol / L. The specificity of the self-made antibody was tested using the expressed normal EGFR ligand binding domain protein and the results showed that the antibody did not bind to the normal EGFR ligand binding domain protein. Conclusion: The anti-mouse EGFRvⅢ monoclonal antibody was successfully prepared, which laid the foundation for the subsequent anti-tumor targeted therapy.