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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∶4.096×106;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 mouse anti-human fibroblast growth factor (hFGF) -21 monoclonal antibody (mAb) and determine its antigenic epitope by bacterial display. Methods The hybridoma cell line secreting anti-human hFGF-21 mAb was screened by hFGF-21 as the detection and immunogen antigen. The mAb was labeled with FITC and the different fragment of hFGF-21 was cloned into the display vector Apex. Cytometer screening of its epitopes. Results A strain of anti-hFGF-21 antibody was successfully screened. The subtype of antibody heavy chain was IgG 2b and the light chain was Kappa chain. The titer of ascites was 1: 4.096 × 106. 30 generations and liquid nitrogen storage for 3 months, the cell strain can stably secrete anti-hFGF-21 mAb, and the titer is stable; Western blot test showed that the antibody has good specificity with human FGF-21; the mAb and Mouse FGF-21 has a cross-reactivity; screening of epitopes by flow cytometry can react with the 107-121 amino acids downstream of hFGF-21. Conclusion The mouse anti-hFGF-21 mAb with high specificity and specificity was successfully prepared, and the epitope of the mAb was identified as amino acids 107-121.