摘要
目的 制备小鼠抗人成纤维细胞生长因子(hFGF)-21单克隆抗体(mAb), 通过细菌展示确定该mAb的抗原表位。方法 用hFGF-21作为检测和免疫抗原, 间接ELISA筛选分泌抗人hFGF-21 mAb的杂交瘤细胞株; 用FITC标记该mAb, 并克隆hFGF-21的不同片段到展示载体Apex上, 通过流式细胞仪筛选其抗原表位。结果 成功筛选出1株抗hFGF-21抗体的细胞株, 其分泌抗体重链的亚型为IgG 2b, 轻链为Kappa链; 该杂交瘤细胞株腹水的效价为1∶4.096×10^6; 传30代及液氮中保存3个月, 该细胞株能稳定分泌抗hFGF-21 mAb, 且效价稳定; Western blot法检测证明该抗体与人FGF-21有很好的特异性; 该mAb与小鼠FGF-21有交叉反应; 通过流式细胞仪对抗原表位的筛选, 该mAb可与hFGF-21下游的第107-121个氨基酸反应。结论 成功的制备出特异性、高稳定性的小鼠抗hFGF-21 mAb, 确定了该mAb的抗原表位在第107~121位氨基酸。
] Objective To prepare monoclonal antibodies (mAbs) against human fibreblast growth factor 21 (hFGF-21), and identify the epitope of hFGF-21 mAb through bacterial display. Methods With hFGF-21 as an immunogen and detective antigen, we screened hybridoma cell lines secreting anti-hFGF-21 mAbs using indirect ELISA. The different fragments of hFGF-21 were cloned into bacterial display vector (Apex) to identify the epitope by fl cell sorting (FACS) using FITC-labeled mAbs. Results We obtained a stable hybridoma cell line secreting mAbs against hFGF-21 ; the heavy and light chains of the mAb were IgG 2b and Kappa, respectively. The ascites titer of the hybridoma cell line was 1:4.096 × 10^6. The cell line was stable after 30 passages or when stored in liquid nitrogen for 3 months. Western blotting showed the mAbs could bind to hFGF-21 specifically, and could cross-react with murine FGF-21. FACS indicated that this antibody could bind to downstream 107-121 amino acids of hFGF-21. Conclusion The mAbs against hFGF-21 we prepared showed high specificity and stability; the epitope of the mAbs was 107-121 amino acids of hFGF-21.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2013年第8期834-837,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
东北农业大学博士启动基金(2010RCB52)