The determination of where an antibody binds on its antigen is the subject of a considerable amount of interest. Here we present the optimization of a set of modeling, cloning, expression and assay techniques to generate a high-throughput scanning mutagenesis platform with wide applicability to epitope mapping programs. A panel of conformation-sensitive, single-chain antibodies against the erythropoietin receptor (EpoR) was mapped. Exhaustive alanine scanning of the receptor surface was combined with a selected set of arginine mutations and competition experiments to provide a relatively complete picture of the epitopes involved. Mutations which significantly decreased or abolished binding were considered to be part of the epitope. The use of matched sets of non-competitive, conformation-sensitive antibodies allowed for the exclusion of false-positives due to gross misfolding of the mutant constructs This platform was then applied to a system where the structure of the protein target, IL-17R, is not known. A major benefit of this technique is that large numbers of different antibody candidates can be mapped at high resolution in parallel. Additionally, the panel of mutant target proteins can also be utilized to generate detailed maps of residue positions in the target protein critical for binding to the native ligand.

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