Patent ID: 7320879
Filing Date: 2008-01-22
Classification: G01N

Abstract:
1. A method of determining the binding site specificity of a first analyte that binds to a ligand having at least two different binding sites, comprising: immobilizing the ligand to a sensing surface of a biosensor, providing a second, reference analyte which binds reversibly to the ligand at a binding site thereof and which is selected to have a dissociation phase, after interaction with the ligand has ceased, that is either substantially faster or substantially slower than that of the first analyte, contacting a mixture of the first analyte and the second, reference analyte with the immobilized ligand to permit association to the ligand, stopping the contacting of the first analyte and the second, reference analyte with the ligand, and subjecting the immobilized ligand to conditions which permit dissociation of bound first analyte and second reference analyte therefrom, monitoring in real time by a label-free detection technique at least the dissociation phase of the interaction of the mixture of the first analyte and the second, reference analyte with the immobilized ligand to obtain a dissociation phase binding curve, successively increasing the concentration in the mixture of the one of the first analyte and the second, reference analyte that has the faster dissociation phase, and determining, from a label-free detection technique, the influence of the increased concentration on the dissociation phase binding curve profile of the mixture to determine therefrom if the first analyte and the second, reference analyte bind to the same or different binding sites on the ligand, wherein an influence in which a substantially reduced contribution to the dissociation phase binding curve profile for the mixture from the one of the first analyte and the second, reference analyte that has the slower dissociation phase indicates that the first analyte and the second, reference analyte bind to the same binding site.