Single-Cell Sorting of HBsAg-Binding Memory B Cells from Human Peripheral Blood Mononuclear Cells and Antibody Cloning

The isolation of human antibodies with naturally paired heavy and light chains is crucial for understanding the human antibody immune response. Here, we present a protocol for antibody cloning from the sorted single human memory B cells recognizing hepatitis B virus (HBV) S antigen (HBsAg). A two-fluorescent-dye labeling strategy against HBsAg allows for an improved sorting specificity, while non-relevant protein staining allows for the exclusion of non-specific B cells. This protocol could also be widely adapted for other antigens. For complete details on the use and execution of this protocol, please refer to Wang et al. (2020). [Display omitted] •Human memory B cells recognizing HBsAg were single-cell sorted into 96-well plates•A two-fluorescent-dye labeling strategy was used to improve the sorting specificity•Three nested PCRs were performed using reverse-transcribed cDNA of sorted single cells•Cloning of naturally paired antibody is critical for studying the human antibody response The isolation of human antibodies with naturally paired heavy and light chains is crucial for understanding the human antibody immune response. Here, we present a protocol for antibody cloning from the sorted single human memory B cells recognizing hepatitis B virus (HBV) S antigen (HBsAg). A two-fluorescent-dye labeling strategy against HBsAg allows for an improved sorting specificity, while non-relevant protein staining allows for the exclusion of non-specific B cells. This protocol could also be widely adapted for other antigens.