An entirely cell-based system to generate single-chain antibodies against cell surface receptors

J Mol Biol. 2008 May 30;379(2):261-72. doi: 10.1016/j.jmb.2008.03.072. Epub 2008 Apr 7.


The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.

Publication types

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

MeSH terms

  • Animals
  • Antibody Affinity
  • Antibody Specificity
  • Biological Assay / methods*
  • Cell Line
  • Drosophila melanogaster / physiology
  • Humans
  • Immunoglobulin Fragments / chemistry*
  • Immunoglobulin Fragments / genetics
  • Immunoglobulin Fragments / metabolism*
  • Immunoglobulin Variable Region / chemistry
  • Immunoglobulin Variable Region / genetics
  • Immunoglobulin Variable Region / metabolism
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Peptide Library
  • Receptors, Cell Surface / chemistry
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Toll-Like Receptor 2 / chemistry
  • Toll-Like Receptor 2 / genetics
  • Toll-Like Receptor 2 / metabolism*


  • Immunoglobulin Fragments
  • Immunoglobulin Variable Region
  • Peptide Library
  • Receptors, Cell Surface
  • Recombinant Proteins
  • Toll-Like Receptor 2