Antibody generation through B cell panning on antigen followed by in situ culture and direct RT-PCR on cells harvested en masse from antigen-positive wells

J Immunol Methods. 2006 Oct 20;316(1-2):133-43. doi: 10.1016/j.jim.2006.08.010. Epub 2006 Sep 18.

Abstract

We describe a method for the generation of high-affinity monoclonal antibodies, which combines the power of natural immune responses with in vitro panning, B cell culture, RT-PCR and expression of the recombinant product. B cells from immunised rabbits were incubated at approximately 1000-10,000 cells per well with solid phase antigen coated on the surface of 96-well ELISA plates. Extensive washing removed non-binding cells as well as those B cells, which bound with low affinity. Retained B cells were cultured for 7 days in the presence of activated rabbit splenocyte supernatant and irradiated EL-4-B5 mouse thymoma cells, to induce proliferation and secretion of immunoglobulin. Supernatants were screened to confirm the presence of specific antibody, before the cells were harvested en masse from individual positive wells. Single heavy- and light-chain variable region genes were recovered from individual wells by RT-PCR, critically without the need for isolation of single B cells. Paired VH and VL genes were subsequently expressed as recombinant antibodies and shown to retain the original activity and specificity of the B cell culture supernatants. The method has also been successfully applied to the generation of high-affinity antibodies to antigen expressed on the surface of target cells.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / biosynthesis*
  • Antibodies, Monoclonal / genetics
  • B-Lymphocytes / immunology*
  • CHO Cells
  • Clone Cells / immunology
  • Cloning, Molecular
  • Cricetinae
  • Enzyme-Linked Immunosorbent Assay
  • Immunoglobulin Heavy Chains / genetics
  • Immunoglobulin Heavy Chains / immunology
  • Immunoglobulin Light Chains / genetics
  • Immunoglobulin Light Chains / immunology
  • Immunoglobulin Variable Region / genetics
  • Immunoglobulin Variable Region / immunology
  • Molecular Sequence Data
  • OX40 Ligand / immunology*
  • RNA / chemistry
  • RNA / genetics
  • Rabbits
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Reverse Transcriptase Polymerase Chain Reaction
  • Surface Plasmon Resonance

Substances

  • Antibodies, Monoclonal
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Light Chains
  • Immunoglobulin Variable Region
  • OX40 Ligand
  • Recombinant Proteins
  • RNA