Identification of recombinant antibodies against multiple distinct toll-like receptors by homolog mining a single immune scFv phage library

J Immunol Methods. 2009 Jan 30;340(2):144-53. doi: 10.1016/j.jim.2008.10.013. Epub 2008 Nov 17.


The generation of recombinant single-chain antibodies from either non-immune or immune phage display antibody libraries is an effective means to obtain high affinity antibodies against a specific target. Non-immune libraries contain a wide variety of antibodies but these are often low affinity. Immune libraries contain a high frequency of high affinity antibodies, but are typically limited to a single antigen. Due to the V(H) and V(L) recombination that occurs during antibody library construction, we hypothesized that an immune antibody library produced against one member of a protein family would contain antibodies specific for other members of the same protein family. Here, we tested this hypothesis by mining an existing anti-human Toll-like receptor-2 (hTLR2) antibody library for antibodies specific for other members of the TLR family. This procedure, referred to as homolog mining, proved to be effective. Using a cell-based system to pan and screen the anti-TLR2 library, we identified single chain antibodies specific for three of the four hTLR2 homologs we targeted. The antibodies identified, anti-murine TLR2, anti-hTLR5, and anti-hTLR6, bind specifically to their target, with no cross-reactivity to hTLR2 or other TLRs tested. These results demonstrate that combinatorial re-assortment of V(H) and V(L) fragments from multiple sources during Ab library construction increases Ab repertoire complexity, allowing antibody libraries produced by immunization with one antigen to be used to obtain antibodies specific to related antigens. The principle of homolog mining may be extended to other protein families and will facilitate and accelerate antibody production processes.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antibodies / analysis*
  • Antibodies / genetics
  • Antibodies / immunology*
  • Antibodies / metabolism
  • Antibody Specificity / immunology
  • Cells, Cultured
  • Humans
  • Immunoglobulin Variable Region / analysis*
  • Immunoglobulin Variable Region / genetics
  • Immunoglobulin Variable Region / immunology*
  • Immunoglobulin Variable Region / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Peptide Library*
  • Recombinant Proteins / analysis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism
  • Toll-Like Receptors / immunology*


  • Antibodies
  • Immunoglobulin Variable Region
  • Peptide Library
  • Recombinant Proteins
  • Toll-Like Receptors