Effect of microsphere binding site density on the apparent affinity of an interaction partner

Background: Flow cytometric microsphere-based binding assays can be used to measure molecular interactions with high sensitivity. We have used multiplexed microsphere technology to explore the effect that binding site density has on the apparent affinity of a soluble interaction partner. Methods: The interaction of a nuclear receptor, peroxisome proliferator-activated receptor gamma ligand binding domain (PPARγ LBD), with a synthetic peptide derived from a nuclear receptor coactivator protein, PPARγ coactivator-1 alpha (PGC-1α), is the interacting system being studied. The density of this peptide coupled to fluorescently unique microsphere populations is varied by co-incubating the biotinylated peptide and avidin-coated microsphere populations with increasing the amounts of free D-biotin. The discrete-density peptide-coupled microsphere populations are combined to conduct a multiplexed binding experiment with Alexa 532-labeled PPARγ LBD, in the absence or presence of a small molecule ligand. Results: As the immobilized binding site density of PGC-1α peptide on fluorescent microspheres is increased the measured apparent affinity for PPARγ LBD is increased. Conclusions: The density of binding sites immobilized to a surface has a pronounced effect on the apparent affinity for soluble binding partners. By controlling and varying the binding site density it is possible to increase the sensitivity of an interaction assay. In multiplexed assay formats it should be possible to normalize intrinsically unequal binding interactions by individually optimizing the binding site density of the immobilized interaction partner. However, to quantitatively measure intrinsic affinities of molecular interactions, low binding site densities are required and multivalent reagents must be avoided. © 2006 International Society for Analytical Cytology