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

Hjelm B, Forsström B, Löfblom J, Rockberg J, Uhlén M - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden.

ABSTRACT
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.

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The three-dimensional structure of the epitopes.Crystal or solution structures of antigens with highlighted antigenic determinants common in all three immunizations determined by bead array mapping in blue. The molecular surface of the protein is colored grey and the antigen part is highlighted in white. The images were acquired using MacPyMOL. Structures of (A) RRM domain of HNRNPH2 (1wez.pdb), (B) PDZ domain of SYNJ2BP (2eno.pdb), (C) PDXP (2cfs.pdb). (D) extracellular domain of ERBB2 (1n8z.pdb). (E) Crystal structure of homodimer TYMP (2jof.pdb) showing the molecular surface with indicated epitopes (blue, green, pink, cyan, orange, yellow and purple) used for affinity purification of monospecific antibodies. (F), View of monomeric TYMP showing secondary structural features of epitopes.
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pone-0045817-g006: The three-dimensional structure of the epitopes.Crystal or solution structures of antigens with highlighted antigenic determinants common in all three immunizations determined by bead array mapping in blue. The molecular surface of the protein is colored grey and the antigen part is highlighted in white. The images were acquired using MacPyMOL. Structures of (A) RRM domain of HNRNPH2 (1wez.pdb), (B) PDZ domain of SYNJ2BP (2eno.pdb), (C) PDXP (2cfs.pdb). (D) extracellular domain of ERBB2 (1n8z.pdb). (E) Crystal structure of homodimer TYMP (2jof.pdb) showing the molecular surface with indicated epitopes (blue, green, pink, cyan, orange, yellow and purple) used for affinity purification of monospecific antibodies. (F), View of monomeric TYMP showing secondary structural features of epitopes.

Mentions: The three-dimensional structures of some of the proteins used for design of antigens are shown in Figure 6. For the targets HNRNPH2 (A), SYNJ2BP (B), PDXP (C) and ERBB2 (D), the epitopes are shown in blue with the “epitope silent” parts of the antigen shown in white. For all four protein targets, the antibodies are directed to epitopes mainly located on the surface of the respective protein target. The epitopes of the antibodies towards TYMP (E) and (F) are also located on the surface the homodimer protein with some parts protruding into the core of the structure. Four of the epitopes are situated on the surface (blue, green, pink and cyan), one partially buried (purple) and two overlapping packed inside the protein (orange and yellow).


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

Hjelm B, Forsström B, Löfblom J, Rockberg J, Uhlén M - PLoS ONE (2012)

The three-dimensional structure of the epitopes.Crystal or solution structures of antigens with highlighted antigenic determinants common in all three immunizations determined by bead array mapping in blue. The molecular surface of the protein is colored grey and the antigen part is highlighted in white. The images were acquired using MacPyMOL. Structures of (A) RRM domain of HNRNPH2 (1wez.pdb), (B) PDZ domain of SYNJ2BP (2eno.pdb), (C) PDXP (2cfs.pdb). (D) extracellular domain of ERBB2 (1n8z.pdb). (E) Crystal structure of homodimer TYMP (2jof.pdb) showing the molecular surface with indicated epitopes (blue, green, pink, cyan, orange, yellow and purple) used for affinity purification of monospecific antibodies. (F), View of monomeric TYMP showing secondary structural features of epitopes.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526615&req=5

pone-0045817-g006: The three-dimensional structure of the epitopes.Crystal or solution structures of antigens with highlighted antigenic determinants common in all three immunizations determined by bead array mapping in blue. The molecular surface of the protein is colored grey and the antigen part is highlighted in white. The images were acquired using MacPyMOL. Structures of (A) RRM domain of HNRNPH2 (1wez.pdb), (B) PDZ domain of SYNJ2BP (2eno.pdb), (C) PDXP (2cfs.pdb). (D) extracellular domain of ERBB2 (1n8z.pdb). (E) Crystal structure of homodimer TYMP (2jof.pdb) showing the molecular surface with indicated epitopes (blue, green, pink, cyan, orange, yellow and purple) used for affinity purification of monospecific antibodies. (F), View of monomeric TYMP showing secondary structural features of epitopes.
Mentions: The three-dimensional structures of some of the proteins used for design of antigens are shown in Figure 6. For the targets HNRNPH2 (A), SYNJ2BP (B), PDXP (C) and ERBB2 (D), the epitopes are shown in blue with the “epitope silent” parts of the antigen shown in white. For all four protein targets, the antibodies are directed to epitopes mainly located on the surface of the respective protein target. The epitopes of the antibodies towards TYMP (E) and (F) are also located on the surface the homodimer protein with some parts protruding into the core of the structure. Four of the epitopes are situated on the surface (blue, green, pink and cyan), one partially buried (purple) and two overlapping packed inside the protein (orange and yellow).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden.

ABSTRACT
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.

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