[Preparation and characterization of monoclonal antibodies against EGFP].

内容摘要

To prepare monoclonal antibodies (mAbs) with high specificity and affinity against enhanced green fluorescent protein (EGFP), we used purified EGFP emulsified with Freund{L-End} 's adjuvant to immunize Balb/c mice. Mouse splenocytes were then fused with SP2/0 myeloma cells. Hybridoma cell lines stably secreting antibodies were obtained through indirect ELISA screening and subcloning with the limited dilution method. The epitope specificity, reactivity, and affinity of the antibodies were systematically analyzed via an additivity assay, isotype identification, Western blotting, and surface plasmon resonance (SPR). The results demonstrated the successful screening of two hybridoma cell lines, 1F11 and 3C11, which stably secreted antibodies specifically targeting EGFP, with isotypes of IgG2a and IgG2b, respectively. The additivity assay showed an additivity index (AI)>40%, indicating recognition of distinct antigenic epitopes. Antibody titers reached 1:512 000. Western blotting confirmed that the prepared antibodies could specifically recognize EGFP with a size of about 27 kDa. SPR analysis revealed affinity constants of 3.806×10-10 mol/L and 2.631×10-10 mol/L, respectively, both within the high-affinity range. These findings indicate that the prepared mAbs possess excellent specificity, reactivity, and high affinity, providing a specific and important biological tool for the immunological detection of EGFP-tagged proteins and the in vivo monitoring of vaccine strains. 为制备高特异性和亲和力的EGFP单克隆抗体(monoclonal antibody, mAb)，本研究以纯化的EGFP蛋白为抗原，经弗氏佐剂乳化后免疫Balb/c小鼠，将小鼠脾细胞与SP2/0骨髓瘤细胞进行融合，应用间接ELISA方法筛选阳性孔并利用有限稀释法进行亚克隆，获得稳定分泌抗体的杂交瘤细胞株。采用叠加试验、亚型鉴定、Western blotting及表面等离子体共振(surface plasmon resonance, SPR)技术，系统分析抗体的表位特异性、反应性及亲和力。结果发现，本研究成功筛选出2株能稳定分泌特异性针对EGFP蛋白单抗的杂交瘤细胞株1F11和3C11，其亚型分别为IgG2a和IgG2b;叠加试验显示叠加系数(additivity index, AI)大于40%，说明两者识别不同抗原表位;抗体效价检测发现效价均可达1:512 000;Western blotting分析证实其可特异性识别大小约为27 kDa的EGFP蛋白;SPR分析测得亲和力常数分别为3.806×10-10 mol/L和2.631×10-10 mol/L，均属于高亲和力水平。由此可见，本研究制备的mAbs具有良好的特异性、反应性及高亲和力，为EGFP标记蛋白的免疫学检测及疫苗株体内监测提供了特异性的重要生物工具。.