Binding Characterization of Cyclic Peptide Ligands to Target Proteins and Chemical Epitopes Using ELISA and Fluorescence Polarization Assays.

Enzyme-linked immunosorbent assay (ELISA) is a plate-based immunological assay designed to detect and quantify peptides, proteins, antibodies, and hormones. Fluorescence polarization (FP) is a solution-phase technique that can be used to determine equilibrium dissociation constant of ligand for the protein of interest. Here we describe the protocols for different ELISAs and for Fluorescence Polarization, and how they can be used to determine relative or absolute binding of macrocyclic peptides to the target proteins. In ELISA, the target protein is used as the antigen, and the binding of antigen is quantified using cyclic peptides and enzyme-linked antibodies. In Fluorescence Polarization assays, a cyclic ligand is fluorescent dye-labeled and titrated with serial concentrations of the non-labeled target protein to determine the equilibrium dissociation constant (KD) of ligand for protein. Detailed descriptions of sample preparation and the ELISA and FP experiments are provided in this chapter.