DETECTION OF LOW-AFFINITY INTERACTIONS BETWEEN PEPTIDES AND HEAT-SHOCK PROTEINS BY CHEMILUMINESCENCE OF ENHANCED AVIDITY REACTIONS (CLEAR)

Protein-protein and protein-peptide interactions that are low affinity in nature preclude the straightforward measurement of binding. To ove rcome this limitation, a novel method has been devised for stabilizing these weak interactions by increasing the binding avidity. These stud ies have focused on the binding of peptides to heat shock proteins (wi th a typical K-D of approximately 25 to 50 mu M). Multivalent ligands have been created by coupling peptides plus biotin to a neutral carrie r molecule, dextran. These peptide-dextran conjugates allow for more a vid binding to proteins that have been immobilized on a membrane surfa ce. Detection of signals via enhanced chemiluminescence further increa ses the sensitivity of the method that has been termed Chemiluminescen ce of Enhanced Avidity Reactions (CLEAR), The assay is simple, reliabl e and consistently detects specific binding between heat shock protein s and peptide ligands. CLEAR should be generally applicable to other l igand receptor pairs where the detection of binding is limited by the low affinity of the interaction.