摘要
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 of EGFRvⅢ Monoclonal Antibody,and it is expected to be good to the tumor＇s target treatment.METHODS：The peptide of EGFRvⅢ was synthesized,and conjugated with KLH.Balb/c mouse was immuned,hybridoma cell was used to generate ascites,antibody from ascites was purified and the speciality of the monoclonal antibody was determined.RESULTS： The corresponding 14-amino acid peptide of the gene absence fusing area of EGFRvⅢ,and conjugatd with KLH.Immune Balb/c mouse using this conjugation as antigen with a certain immune protocal,when high titer antiserum（1∶128 000）was obtained,fuse the spleen cell of immuned mouse with SP2/0 cell,select by ELISA and subclone to obtain anti EGFRvⅢ stabilized hybridoma cell lines.5 mouse hybridoma lines obtained and ascites produced.The type of Monoclonal Antibody is IgG2a,Determine ascites antibody titer and its affinity with antigen.The titer was 1∶128 000,and the highest affinity of those was 9.8×10-9 mol/L.The speciality of the monoclonal antibody was determined by the expressed natural EGFRs binding domain in Pichia yeast.The results showed that the antibody could not bind with the expressed peptide.It indicated that the monoclonal antibody prepared by ourselves was special to EGFRvⅢ.CONCLUSION： The succeeding preparation of EGFRvⅢ monoclonal antibody by this experiment gives a stability base to the tumor＇s target treatment.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2012年第3期192-196,共5页
Chinese Journal of Cancer Prevention and Treatment
