Parallel immunizations of rabbits using the same antigen yield antibodies with similar, but not identical, epitopes

PLoS One. 2012;7(12):e45817. doi: 10.1371/journal.pone.0045817. Epub 2012 Dec 19.


A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies / immunology*
  • Antibody Specificity*
  • Blotting, Western
  • Epitope Mapping
  • Epitopes / chemistry
  • Epitopes / immunology*
  • HEK293 Cells
  • Humans
  • Immunization*
  • Models, Molecular
  • Peptide Fragments / immunology
  • Protein Array Analysis
  • Protein Conformation
  • Proteome / immunology*
  • Proteomics
  • Rabbits


  • Antibodies
  • Epitopes
  • Peptide Fragments
  • Proteome

Grants and funding

This work was supported by grants from the Knut and Alice Wallenberg Foundation, Swedish Research Council and the VINNOVA ProNova center grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.