摘要
目的探讨流式探针法在分析、分选丙型肝炎病毒(hepatitis C virus, HCV)特异性B细胞以及全人源单克隆抗体发现中的作用。方法采用伯胺标记法制备HCV特异性探针;使用流式细胞术检测和分选HCV特异B细胞,并体外通过单个B细胞抗体克隆技术从分选的HCV特异性B细胞中克隆表达抗体;应用ELISA技术和假病毒中和试验检测抗体的结合活性以及中和活性。结果探针法能有效地检测HCV特异的记忆性B细胞,HCV患者探针阳性细胞频率明显高于正常人(U=0.0004,P <0.0001)。通过单细胞分选HCV特异的记忆性B细胞,体外克隆表达获得6个单克隆抗体,其中有5个与HCV膜蛋白具有结合活性,2个显示具有较强中和活性。结论伯氨标记法制备HCV特异性探针是一种简单高效的探针制备方法,结合流式细胞术可用于HCV特异B细胞的检测以及分选。
Objective To explore the role of probe fluorescence in analysis and sorting of hepatitis C virus(HCV)-specific B cells and discover fully human monoclonal antibodies.Methods The primary amine labeling method was used to prepare HCV-specific probes.HCV-specific B cells were analyzed and sorted by flow cytometry.Single B cell antibody technology was applied to clone antibodies in vitro.Enzyme-linked immunosorbent assay(ELISA)and pseudovirus neutralization assay were performed for the detection of antibody binding and netralization activity.Results The frequency of HCV probe positive cells(0.385±0.17%)in memory B cells of patients with chronic HCV infection was significantly higher than that of healthy subjects(0.012±0.02%)(P<0.01).In addition,6 monoclonal antibodies were cloned,5 of these antibodies showed binding activity with HCV E2c protein,2 of them showed neutralizing response to HCV pseudotyped virus.Conclusions The primary ammonia labeling method is a simple and efficient method for preparing HCV-specific probes,combination of which with flow cytometry was useful for the analysis and sorting of HCV-specific B cells.
作者
牛玲
胡雅彬
伍谦
张健
刘文培
Niu Ling;Hu Yabin;Wu Qian;Zhang Jian;Liu Wenpei(Chenzhou Hospital Affiliated to University of South China,Chenzhou 423000,China;Translational Medicine Institute,University of South China,Chenzhou 423000,China)
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2020年第6期649-654,共6页
Chinese Journal of Experimental and Clinical Virology
基金
郴州市第一人民医院院内重点项目(N2019-002)
郴州市科技局一般课题(zdyf201920,zdyf201921)
郴州市第一人民医院优秀青年基金(N2020-4)。
