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Dissecting antibodies with regards to linear and conformational epitopes.

Forsström B, Axnäs BB, Rockberg J, Danielsson H, Bohlin A, Uhlen M - PLoS ONE (2015)

Bottom Line: An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes.In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays.The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.

View Article: PubMed Central - PubMed

Affiliation: Science for Life Laboratory, KTH-Royal Institute of Technology, SE-171 21 Stockholm, Sweden.

ABSTRACT
An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.

No MeSH data available.


Proportion of antibodies towards linear and conformational epitopes in eight polyclonal sera.Each protein target is represented in grey with the amino acid length indicated under the bar. Antigens used for immunization are shown above in green with the amino acid lengths shown above. Pie charts to the right of each protein show the relative amount of purified antibodies toward conformational (light blue) and linear (dark blue) epitopes.
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pone.0121673.g002: Proportion of antibodies towards linear and conformational epitopes in eight polyclonal sera.Each protein target is represented in grey with the amino acid length indicated under the bar. Antigens used for immunization are shown above in green with the amino acid lengths shown above. Pie charts to the right of each protein show the relative amount of purified antibodies toward conformational (light blue) and linear (dark blue) epitopes.

Mentions: We decided to investigate eight polyclonal antibodies generated towards human recombinant protein fragment (PrESTs) with the lengths of the eight antigens varying between 95–149 amino acids. The sizes (number of amino acids) and locations sizes for the eight antigens on their respective full-length protein sequence are shown in Fig 2 together with pie charts showing the fractions antibodies targeting linear and conformational epitopes in the polyclonal sera. For six of the protein targets (OTC, TYMP, CRABP2, PDXP, CD4 and EGFR), a large majority (70–90%) of the antibodies recognize linear epitopes, while for one of the targets (SYNJ2BP) the majority (80%) of the antibodies are shown to bind conformational epitopes. For WARS, already described above, approximately 50% of the antibodies are linear and conformational, respectively. The results suggest that most of the epitopes generated in this manner yield antibodies recognizing linear epitopes.


Dissecting antibodies with regards to linear and conformational epitopes.

Forsström B, Axnäs BB, Rockberg J, Danielsson H, Bohlin A, Uhlen M - PLoS ONE (2015)

Proportion of antibodies towards linear and conformational epitopes in eight polyclonal sera.Each protein target is represented in grey with the amino acid length indicated under the bar. Antigens used for immunization are shown above in green with the amino acid lengths shown above. Pie charts to the right of each protein show the relative amount of purified antibodies toward conformational (light blue) and linear (dark blue) epitopes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4376703&req=5

pone.0121673.g002: Proportion of antibodies towards linear and conformational epitopes in eight polyclonal sera.Each protein target is represented in grey with the amino acid length indicated under the bar. Antigens used for immunization are shown above in green with the amino acid lengths shown above. Pie charts to the right of each protein show the relative amount of purified antibodies toward conformational (light blue) and linear (dark blue) epitopes.
Mentions: We decided to investigate eight polyclonal antibodies generated towards human recombinant protein fragment (PrESTs) with the lengths of the eight antigens varying between 95–149 amino acids. The sizes (number of amino acids) and locations sizes for the eight antigens on their respective full-length protein sequence are shown in Fig 2 together with pie charts showing the fractions antibodies targeting linear and conformational epitopes in the polyclonal sera. For six of the protein targets (OTC, TYMP, CRABP2, PDXP, CD4 and EGFR), a large majority (70–90%) of the antibodies recognize linear epitopes, while for one of the targets (SYNJ2BP) the majority (80%) of the antibodies are shown to bind conformational epitopes. For WARS, already described above, approximately 50% of the antibodies are linear and conformational, respectively. The results suggest that most of the epitopes generated in this manner yield antibodies recognizing linear epitopes.

Bottom Line: An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes.In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays.The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.

View Article: PubMed Central - PubMed

Affiliation: Science for Life Laboratory, KTH-Royal Institute of Technology, SE-171 21 Stockholm, Sweden.

ABSTRACT
An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.

No MeSH data available.