A rapid and scalable method for selecting recombinant mouse monoclonal antibodies

BMC Biol. 2010 Jun 4;8:76. doi: 10.1186/1741-7007-8-76.

Abstract

Background: Monoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas.

Results: Increased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos.

Conclusions: This method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.

Publication types

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

MeSH terms

  • Animals
  • Antibodies, Monoclonal / genetics*
  • Antibodies, Monoclonal / immunology
  • Antibodies, Monoclonal / isolation & purification
  • Cell Line
  • Cell Line, Tumor
  • Cloning, Molecular / methods*
  • Gene Expression
  • Humans
  • Hybridomas / immunology
  • Immunoglobulin G / immunology
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / immunology
  • Recombinant Proteins / isolation & purification
  • Zebrafish / embryology
  • Zebrafish / immunology

Substances

  • Antibodies, Monoclonal
  • Immunoglobulin G
  • Recombinant Proteins