Chemistry

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

Document Type

Book Chapter

Abstract

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.

Publication Title

Methods in Molecular Biology

Publication Date

2022

Volume

2371

First Page

335

Last Page

354

ISSN

1064-3745

DOI

10.1007/978-1-0716-1689-5_18

Keywords

chemical epitope, enzyme-linked immunosorbent assay, Fluorescence polarization, macrocyclic peptides, protein–peptide binding

Cross Post Location

Student Publications

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