摘要
目的:制备并鉴定特异性抗人N端脑钠肽前体(NT-proBNP)抗原表位的单克隆抗体。方法:通过Bepipred在线表位预测方法和Epitope Prediction System软件预测人NT-proBNP B细胞抗原表位,综合2种预测结果对比优化选取合适的2个表位肽段P1和P2,偶联KLH为免疫原,免疫BALB/c小鼠,选择血清效价较高的小鼠脾细胞与SP2/0细胞融合;经HAT筛选,间接ELISA法检测和有限稀释法进行亚克隆,得到阳性杂交瘤细胞株;采用体内诱生法制备腹水并用Protein G柱纯化抗体;经鉴定,对特异性最好的2株进行纯度、效价、亚型及亲和力的分析。结果:通过P2-KLH免疫获得的阳性杂交瘤细胞株1F9和P1-KLH免疫获得的阳性杂交瘤细胞株4H2能稳定分泌抗NT-proBNP单克隆抗体,纯度较高,能与NT-proBNP重组蛋白特异性结合,效价分别达到4×10^4、4×10^5,抗体亚型均为IgG1,相对亲和力常数分别为1.14×10^7和1.6×10^7 L/mol。结论:获得2株特性良好的单克隆抗体,可为下一步研制NT-proBNP检测试剂盒提供原材料。
Objective:To prepare and identify specific anti-human N-terminal pro-brain natriuretic peptide(NTproBNP)epitope monoclonal antibody.Methods:The Bepipred online epitope prediction and the Epitope Prediction System software were used to predict the NT-proBNP B cell epitope,and the two prediction results were compared and optimized to select the appropriate two epitope peptides.This experiment used two peptides predicted which are coupling carrier protein KLH as immunogens.After immunization of BALB/c mice,spleen cells of mice with the higher serum titer were selected to fuse with SP2/0 cells.HAT screening,indirect ELISA detection and limiting dilution method were used for subcloning to obtain positive hybridoma cell lines.Ascites was prepared by induction in vivo and antibodies were purified by Protein G column.After identifications,two highest specific strains were selected for the analysis of purity,titer,subtype and affinity.Results:Positive hybridoma cell line 1F9 obtained by immunization with P2-KLH and 4H2 obtained by P1-KLH both secreted anti-NT-proBNP mono762 clonal antibodies with high purities and specifically bound to NT-proBNP recombinant protein.The titers reached 4×10^4,4×10^5 respectively.The subtypes were both IgG1.The Ka were 1.14×10^7,1.6×10^7 L/mol.Conclusion:The results showed that the two monoclonal antibodies had good properties and could be used as test materials for the next step of developing the NT-proBNP detection kit.
作者
侯亚璐
井金苗
魏治静
柳峰松
吴萌
HOU Ya-Lu;JING Jin-Miao;WEI Zhi-Jing;LIU Feng-Song;WU Meng(College of Life Science,Hebei University,Baoding 071002;Biology Institute,Hebei Academy of Sciences,Shijiazhuang 050081,China)
出处
《生物技术通讯》
CAS
2019年第6期762-767,共6页
Letters in Biotechnology
基金
河北省高层次人才资助项目（E2018050016）
关键词
人N端脑钠肽前体
B细胞抗原表位
单克隆抗体
间接ELISA法
抗体特性
human N-terminal pro-brain natriuretic peptide(NT-proBNP)
B cell epitope
monoclonal antibody
indirect ELISA method
antibody characteristic
