A robust high throughput platform to generate functional recombinant monoclonal antibodies using rabbit B cells from peripheral blood

PLoS One. 2014 Feb 4;9(2):e86184. doi: 10.1371/journal.pone.0086184. eCollection 2014.

Abstract

We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / biosynthesis*
  • Antibodies, Monoclonal / blood*
  • Antibody Specificity / immunology
  • B-Lymphocytes / metabolism*
  • Cell Separation
  • Cells, Cultured
  • Clone Cells
  • Epitopes / immunology
  • HEK293 Cells
  • High-Throughput Screening Assays / methods*
  • Humans
  • Immunization
  • Immunoglobulin Heavy Chains / immunology
  • Immunoglobulin Light Chains / immunology
  • Immunoglobulin Variable Region / immunology
  • Interleukin-1 Receptor-Like 1 Protein
  • Interleukin-33
  • Interleukins / metabolism
  • Protein Binding
  • Rabbits
  • Receptors, Cell Surface / metabolism
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / blood*

Substances

  • Antibodies, Monoclonal
  • Epitopes
  • IL1RL1 protein, human
  • IL33 protein, human
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Light Chains
  • Immunoglobulin Variable Region
  • Interleukin-1 Receptor-Like 1 Protein
  • Interleukin-33
  • Interleukins
  • Receptors, Cell Surface
  • Recombinant Proteins

Grant support

All work presented here was funded by the commercial company Roche. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.