人成纤维细胞生长因子-21单克隆抗体的制备及抗原表位的确定

Preparation of monoclonal antibodies against hFGF-21 and identification of epitope

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.