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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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Schematic overview of the epitope mapping methods used in the study.(A) Epitope mapping using bacterial display, in which the target gene is fragmented and a library of clones is expressed on S. carnosus. The cell displayed peptide library is assayed for binding to the antibody using a flow cytometer Binding clones are sorted, sequenced and aligned back to the antigen sequence in order to conclude epitopes. (B) The sequencing of the flow-sorted libraries is used to determine the epitope regions. To the left, a typical FACS dot plot showing sorting of a cell displayed library incubated with the investigated antibody. The colored bars to the right show aligned binding sequences from the different sorted populations and on top the consensus epitopes derived from the alignment. (C) Epitope mapping using peptide bead arrays with overlapping peptides, spanning the antigen sequence, coupled to color coded beads. Antibody binding towards the peptides is evaluated using a flow cytometer instrument and epitope regions are identified on the antigen. (D) A schematic view of how binding profiles are used to determine epitope regions.
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pone-0045817-g001: Schematic overview of the epitope mapping methods used in the study.(A) Epitope mapping using bacterial display, in which the target gene is fragmented and a library of clones is expressed on S. carnosus. The cell displayed peptide library is assayed for binding to the antibody using a flow cytometer Binding clones are sorted, sequenced and aligned back to the antigen sequence in order to conclude epitopes. (B) The sequencing of the flow-sorted libraries is used to determine the epitope regions. To the left, a typical FACS dot plot showing sorting of a cell displayed library incubated with the investigated antibody. The colored bars to the right show aligned binding sequences from the different sorted populations and on top the consensus epitopes derived from the alignment. (C) Epitope mapping using peptide bead arrays with overlapping peptides, spanning the antigen sequence, coupled to color coded beads. Antibody binding towards the peptides is evaluated using a flow cytometer instrument and epitope regions are identified on the antigen. (D) A schematic view of how binding profiles are used to determine epitope regions.

Mentions: Established methods for epitope mapping of antibodies involves chemical synthesis of peptides [13], [14] or peptide display on phages [15], [16]. Recently, we have described two independent methods for epitope mapping of antibodies [17], as schematically outlined in Figure 1. The first method relies on bacterial surface display on Staphylococcus carnosus in which the gene encoding the target protein is fragmented, cloned into an expression vector and subsequently introduced into S. carnosus host cells (Figure 1A). A library of bacterial cells is created, each member with a small fragment of the original gene expressed on the surface of the cell. The cells are incubated with the antibody to be mapped labeled with a fluorescent dye and the cells are analyzed in a flow cytometer so that cells expressing fragments bound by the antibody can be collected. These cells are grown, the insert of the expression vector DNA sequenced and the insert is mapped back to the original gene sequence. In this way, the amino acid sequence binding to the antibody can be mapped back in an efficient manner. The second method relies on suspension bead arrays with color-coded beads, in which each has a synthetic peptide bound to its surface (Figure 1C). The bead mixture with overlapping peptides spanning the whole antigen sequence is incubated with the fluorescently labeled antibody and the beads are analyzed on a flow sorter capable of identifying each color-coded bead. Both methods will give an “apparent affinity” of the binding to the corresponding epitope, i.e. a signal corresponding to the amount of bound antibody. However, it should be noted that since the antibody is polyclonal, the signal is dictated both by the affinities as well as the amount of antibodies directed to the particular epitope in the polyclonal mixture. The latter method relies on binding to linear epitopes, while the bacterial surface display method should be able to identify longer structural epitopes.


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)

Schematic overview of the epitope mapping methods used in the study.(A) Epitope mapping using bacterial display, in which the target gene is fragmented and a library of clones is expressed on S. carnosus. The cell displayed peptide library is assayed for binding to the antibody using a flow cytometer Binding clones are sorted, sequenced and aligned back to the antigen sequence in order to conclude epitopes. (B) The sequencing of the flow-sorted libraries is used to determine the epitope regions. To the left, a typical FACS dot plot showing sorting of a cell displayed library incubated with the investigated antibody. The colored bars to the right show aligned binding sequences from the different sorted populations and on top the consensus epitopes derived from the alignment. (C) Epitope mapping using peptide bead arrays with overlapping peptides, spanning the antigen sequence, coupled to color coded beads. Antibody binding towards the peptides is evaluated using a flow cytometer instrument and epitope regions are identified on the antigen. (D) A schematic view of how binding profiles are used to determine epitope regions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526615&req=5

pone-0045817-g001: Schematic overview of the epitope mapping methods used in the study.(A) Epitope mapping using bacterial display, in which the target gene is fragmented and a library of clones is expressed on S. carnosus. The cell displayed peptide library is assayed for binding to the antibody using a flow cytometer Binding clones are sorted, sequenced and aligned back to the antigen sequence in order to conclude epitopes. (B) The sequencing of the flow-sorted libraries is used to determine the epitope regions. To the left, a typical FACS dot plot showing sorting of a cell displayed library incubated with the investigated antibody. The colored bars to the right show aligned binding sequences from the different sorted populations and on top the consensus epitopes derived from the alignment. (C) Epitope mapping using peptide bead arrays with overlapping peptides, spanning the antigen sequence, coupled to color coded beads. Antibody binding towards the peptides is evaluated using a flow cytometer instrument and epitope regions are identified on the antigen. (D) A schematic view of how binding profiles are used to determine epitope regions.
Mentions: Established methods for epitope mapping of antibodies involves chemical synthesis of peptides [13], [14] or peptide display on phages [15], [16]. Recently, we have described two independent methods for epitope mapping of antibodies [17], as schematically outlined in Figure 1. The first method relies on bacterial surface display on Staphylococcus carnosus in which the gene encoding the target protein is fragmented, cloned into an expression vector and subsequently introduced into S. carnosus host cells (Figure 1A). A library of bacterial cells is created, each member with a small fragment of the original gene expressed on the surface of the cell. The cells are incubated with the antibody to be mapped labeled with a fluorescent dye and the cells are analyzed in a flow cytometer so that cells expressing fragments bound by the antibody can be collected. These cells are grown, the insert of the expression vector DNA sequenced and the insert is mapped back to the original gene sequence. In this way, the amino acid sequence binding to the antibody can be mapped back in an efficient manner. The second method relies on suspension bead arrays with color-coded beads, in which each has a synthetic peptide bound to its surface (Figure 1C). The bead mixture with overlapping peptides spanning the whole antigen sequence is incubated with the fluorescently labeled antibody and the beads are analyzed on a flow sorter capable of identifying each color-coded bead. Both methods will give an “apparent affinity” of the binding to the corresponding epitope, i.e. a signal corresponding to the amount of bound antibody. However, it should be noted that since the antibody is polyclonal, the signal is dictated both by the affinities as well as the amount of antibodies directed to the particular epitope in the polyclonal mixture. The latter method relies on binding to linear epitopes, while the bacterial surface display method should be able to identify longer structural epitopes.

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.

Show MeSH
Related in: MedlinePlus