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

STAR Protoc. 2020 Oct 16;1(3):100129. doi: 10.1016/j.xpro.2020.100129. eCollection 2020 Dec 18.

Abstract

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).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • B-Lymphocytes / immunology
  • Cloning, Molecular / methods
  • Flow Cytometry / methods*
  • Hepatitis B / immunology
  • Hepatitis B Antibodies / genetics
  • Hepatitis B Surface Antigens / immunology
  • Hepatitis B Surface Antigens / isolation & purification
  • Hepatitis B Surface Antigens / metabolism
  • Humans
  • Immunoglobulins / metabolism
  • Leukocytes, Mononuclear / cytology
  • Memory B Cells / cytology*

Substances

  • Hepatitis B Antibodies
  • Hepatitis B Surface Antigens
  • Immunoglobulins