Detection of hepatitis C virus-specific B cell and isolation of fully human-originated neutralizing antibody using probe-based fluorescence activated cell sorting
10.3760/cma.j.cn112866-20200916-00246
- VernacularTitle:基于流式探针法的丙型肝炎病毒特异的B细胞检测与全人源中和抗体发现
- Author:
Ling NIU
1
;
Yabin HU
;
Qian WU
;
Jian ZHANG
;
Wenpei LIU
Author Information
1. 南华大学附属郴州医院，郴州 423000
- Keywords:
Hepatitis C virus;
Neutralizing antibody;
Probe;
Vaccine;
Memory B cell
- From:
Chinese Journal of Experimental and Clinical Virology
2020;34(6):649-654
- CountryChina
- Language:Chinese
-
Abstract:
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.
