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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, n 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。n 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%) (n 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.n 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.