title: Ribosome display enhanced by next generation sequencing: a tool to identify antibody-specific peptide ligands.
abstract: Detection of antibodies in serum has many important applications. Our goal was to develop a facile general experimental approach for identifying antibody-specific peptide ligands that could be used as the reagents for antibody detection. Our emphasis was on an approach that would allow identification of peptide ligands for antibodies in serum without the need to isolate the target antibody or to know the identity of its antigen. We combined ribosome display (RD) with the analysis of peptide libraries by next generation sequencing (NGS) of their coding RNA to facilitate identification of antibody-specific peptide ligands from random sequence peptide library. We first demonstrated, using purified antibodies, that with our approach-specific peptide ligands for antibodies with simple linear epitopes, as well as peptide mimotopes for antibodies recognizing complex epitopes, were readily identified. Inclusion of NGS analysis reduced the number of RD selection rounds that were required to identify specific ligands and facilitated discrimination between specific and spurious nonspecific sequences. We then used a model of human serum spiked with a known target antibody to develop NGS-based analysis that allowed identification of specific ligands for a target antibody in the context of an overwhelming amount of unrelated immunoglobins present in serum.
authors: Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, St. Louis, MO 63104, USA.
authors: Ewa
authors: E
authors: Heyduk
authors: Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, St. Louis, MO 63104, USA. Electronic address: heydukt@slu.edu.
authors: Tomasz
authors: T
authors: Heyduk
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry*; D001483:Base Sequence; D017931:DNA Primers; D008024:Ligands; D010455:Peptides / Q000737:chemistry*; D012270:Ribosomes / Q000737:chemistry*; D017421:Sequence Analysis / Q000379:methods*
chemical_list: D000906:Antibodies; D017931:DNA Primers; D008024:Ligands; D010455:Peptides
keywords: Antibody; Ligand; Next generation sequencing; Peptide; Ribosome display