H因子结合蛋白单克隆抗体的制备及ELISA法含量检测初探  

Preparation of monoclonal antibodies against fHBP and its application in determination of antigen content by ELISA

作　　者：苏桂民 冀颖 纪国存 龙静 郭通 申卫家 张宇 朱卫华 杜琳 SU Gui-min;JI Ying;JI Guo-cun;LONG Jing;GUO Tong;SHEN Wei-jia;ZHANG Yu;ZHU Wei-hua;DU Lin(Beijing Bacterial Vaccine Engineering Research Centre,Beijing 100176,China;Beijing Zhifei Lvzhu Biopharmaceutical Co.,Ltd.,Beijing 100176,China)

机构地区：[1]北京市细菌性疫苗工程技术研究中心,北京100176 [2]北京智飞绿竹生物制药有限公司,北京100176

出　　处：《中国新药杂志》2021年第10期921-928,共8页Chinese Journal of New Drugs

摘　　要：目的:制备B群脑膜炎球菌H因子结合蛋白A(fHBP A)和H因子结合蛋白B(fHBP B)亚家族的单克隆抗体,建立检测fHBP抗原含量的ELISA方法。方法:重组表达并纯化fHBP A(His)和fHBP B(His)标签蛋白免疫小鼠,将小鼠脾细胞与SP2/0细胞融合,筛选3株稳定分泌抗fHBP单克隆抗体的细胞株A,B和AB。以抗AB为一抗,辣根过氧化物酶标记抗A、抗B为二抗,分别建立检测fHBP A和fHBP B的双抗夹心ELISA方法。结果:3种单抗效价均在4.4×10^(5)~8.8×10^(5),Western Blot及ELISA显示3种抗体均有结合特异性,且单抗AB和A,B对应不同的抗原识别位点,相互独立。双抗夹心ELISA方法灵敏度达到10 ng·mL^(-1),变异系数<10%。结论:采用单克隆抗体检测fHBP抗原的ELISA方法特异性和适用性良好,为进一步建立快速灵敏检测和鉴定fHBP抗原的方法奠定了基础。Objective:To obtain monoclonal antibodies of the factor H binding protein(fHBP)A and B subfamily proteins in meningococcal group B,and establish an ELISA detection method which can provide a new way for quantitation and identification of fHBP antigen.Methods:Mice were immunized with purified histidine-labeled recombinant fHBP(His),the spleen cells of mouse were fused with SP2/0 cells.3 cell strains with stable secretion of anti-fHBP monoclonal antibodies were acquired,respectively named A,B and AB.To establish a double-antibody sandwich ELISA method for detecting recombinant fHBP antigen,AB was coated as primary antibodies,A and B were labeled by horseradish peroxidase(HRP)as the second antibody.Results:The GMTs valences of A,B and AB antibodies produced in mouse ascites were all between 4.4~8.8×10^(5),and Western Blot showed that the 3 antibodies could specifically bind the corresponding recombinant fHBP antigens.The direct ELISA test also demonstrated that the 3 monoclonal antibodies merely reacted with proteins containing fHBP A or fHBP B through different recognition sites on antigens,independent of each other.The double-antibody sandwich ELISA method had good specificity and applicability,with sensitivity up to 10 ng·mL^(-1) and coefficient variation of less than 10%.Conclusion:The double antibody sandwich ELISA method based on monoclonal antibody has good specificity and applicability,which laid a foundation to further establish a sensitive and rapid method for detection and identification of fHBP antigen.

关 键 词：H因子结合蛋白 单克隆抗体 ELISA B群脑膜炎球菌 

分 类 号：R943.42[医药卫生—药剂学] R979.1[医药卫生—药学]