A Cell-Free, Nonisotopic, High-Throughput Assay for Inhibitors of Type-I Interleukin-1 Receptor

A cell-free, nonisotopic assay has been developed to discover molecules that compete with the natural ligands for binding to the active site of the Type-I interleukin-1 receptor. The key reagents are the interleukin-1 receptor antagonist, a recombinant soluble form of the receptor (sIL-1R), and a specific anti-sIL-1R nonneutralizing monoclonal antibody (MAb79). With these molecules a sensitive assay has been developed using a reversed format: the ligand is immobilized and the receptor is in solution. The ligand-bound receptor is detected using MAb79 and an enzyme-linked secondary antibody. Since no cells or cell membranes are used, the assay is very robust, with no interference from membrane-perturbing agents and high resistance to the organic solvents normally used to resuspend compounds of chemical libraries. The microplate format and colorimetric detection have allowed the complete automation of the immobilized-ligand IL-1 receptor binding assay, which has been used for high-throughput screening of synthetic compounds and natural products.