Sequencing, cloning, and antigen binding analysis of monoclonal antibodies isolated from single mouse B cells

STAR Protoc. 2021 Mar 15;2(2):100389. doi: 10.1016/j.xpro.2021.100389. eCollection 2021 Jun 18.

Abstract

The analysis of B cell receptors (BCR) from single B cells is crucial to understanding humoral immune responses. Here, we describe a protocol for the sequencing, cloning, and characterization of antibody genes that encode BCRs. We used this method to analyze the BCRs of different mouse B cell populations for somatic hypermutations, clonal and phylogenic relationships, and their affinity for cognate antigen. For complete details on the use and execution of this protocol, please refer to Viant et al. (2020).

Keywords: Antibody; Cell isolation; Immunology; Molecular biology; Protein expression and purification; Sequencing; Single cell.

Publication types

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

MeSH terms

  • Animals
  • Antibodies, Monoclonal* / analysis
  • Antibodies, Monoclonal* / chemistry
  • Antibodies, Monoclonal* / genetics
  • Antibodies, Monoclonal* / metabolism
  • Antigens* / analysis
  • Antigens* / chemistry
  • Antigens* / metabolism
  • B-Lymphocytes / chemistry*
  • Cloning, Molecular / methods*
  • Female
  • Male
  • Mice
  • Protein Binding
  • Sequence Analysis, DNA / methods*
  • Single-Cell Analysis / methods*

Substances

  • Antibodies, Monoclonal
  • Antigens