Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells

Nat Biotechnol. 2010 Sep;28(9):965-9. doi: 10.1038/nbt.1673. Epub 2010 Aug 29.

Abstract

Isolation of antigen-specific monoclonal antibodies (mAbs) and antibody fragments relies on high-throughput screening of immortalized B cells or recombinant antibody libraries. We bypassed the screening step by using high-throughput DNA sequencing and bioinformatic analysis to mine antibody variable region (V)-gene repertoires from bone marrow plasma cells (BMPC) of immunized mice. BMPCs, which cannot be immortalized, produce the vast majority of circulating antibodies. We found that the V-gene repertoire of BMPCs becomes highly polarized after immunization, with the most abundant sequences represented at frequencies between approximately 1% and >10% of the total repertoire. We paired the most abundant variable heavy (V(H)) and variable light (V(L)) genes based on their relative frequencies, reconstructed them using automated gene synthesis, and expressed recombinant antibodies in bacteria or mammalian cells. Antibodies generated in this manner from six mice, each immunized with one of three antigens were overwhelmingly antigen specific (21/27 or 78%). Those generated from a mouse with high serum titers had nanomolar binding affinities.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology
  • Antibodies, Monoclonal / isolation & purification*
  • Antigens / immunology
  • Base Sequence
  • Complementarity Determining Regions / immunology
  • High-Throughput Nucleotide Sequencing
  • High-Throughput Screening Assays / methods*
  • Humans
  • Immunization
  • Immunoglobulin Heavy Chains / genetics
  • Immunoglobulin Variable Region / genetics*
  • Mice
  • Molecular Sequence Data
  • Plasma Cells / immunology*

Substances

  • Antibodies, Monoclonal
  • Antigens
  • Complementarity Determining Regions
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Variable Region