Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

MAbs. Mar-Apr 2014;6(2):493-501. doi: 10.4161/mabs.27435. Epub 2013 Dec 11.

Abstract

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

Keywords: CDRH3; data mining; enrichment; high-throughput sequencing; repertoire.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / genetics
  • Antibodies / immunology*
  • Antibody Specificity
  • B-Lymphocytes / immunology*
  • Complementarity Determining Regions
  • Computational Biology
  • DNA, Complementary / analysis
  • High-Throughput Nucleotide Sequencing
  • Immunization
  • Immunoglobulin Heavy Chains
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Muramidase / immunology
  • Nucleic Acid Hybridization / methods*
  • Protein Binding
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology*

Substances

  • Antibodies
  • Complementarity Determining Regions
  • DNA, Complementary
  • Immunoglobulin Heavy Chains
  • Recombinant Proteins
  • hen egg lysozyme
  • Muramidase