Abstract
Detailed knowledge of antigenic determinants is crucial when characterizing therapeutic and diagnostic antibodies, assessing vaccine effectiveness and developing epitope-based vaccines. Most epitope mapping approaches are labor intensive and costly. In this study, we evaluated panning of phage-displayed random peptide libraries against antibodies as a tool for cognate epitope identification. We used six antibodies directed to three model protein antigens as targets to show that the approach is applicable to both mono- and polyclonal antibodies. The technique is well-suited especially for identification of linear epitopes. Mapping of conformational epitopes is more challenging, tends to be more subjective and requires use of computational tools. Nevertheless, when combined with functional data such as structure-activity relationship of antigen muteins, one can make reliable conformational epitope predictions based on phage display experiment data. As the described approach is fast and relatively inexpensive, we suggest it is employed early in antibody characterization and later validated by complementary methods.
Highlights
Proteins generally make good immunogens owing to their structural complexity and relatively high molecular mass
Each target was probed with 2 libraries; one containing random dodecapeptides, and the other comprising conformationally constrained cyclic peptides of the CX7C general structure, where X is any of the 20 proteinogenic amino acids
In panning experiments against the polyclonal antibody AF-398 we enriched a number of peptides that presumably mimic leptin’s discontinuous determinants alongside linear epitope mimetics
Summary
Proteins generally make good immunogens owing to their structural complexity and relatively high molecular mass. Protein-derived B cell antigenic determinants can be fairly large with a surface area of above 1000 Å2.1 only a handful of amino acid residues typically engage into energetically relevant interactions with the antibody binding site, constituting the so called functional epitope.[2] Such an epitope is considered linear if its residues are arranged in sequential fashion. Only linear epitopes can be unambiguously identified in this manner, there are computational approaches that can aid in mapping peptide epitope mimetics to three-dimensional structure of protein antigens.[14,15]. We present data on epitope mapping by random peptide phage library screening of six commercial antibodies (2 monoclonal and 4 polyclonal) directed against three model polypeptides: chemokine CCL2 and adipokine leptin (two proteins with compact fold), and ghrelin, a peptide hormone with poorly defined secondary structure in aqueous solutions. The predicted conformational epitope recognized by the neutralizing antibody against CCL2 agrees well with the data on surface residues crucial for binding to cognate receptor from mutagenesis studies
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