Assay for evaluating affinity and specificity of ligand-albumin binding

A method for determining both the affinity and specificity of a ligand which binds to albumin in a single assay, comprising the steps of:

• a) contacting a reaction mixture comprising a site-specific fluorescent probe and albumin in the presence, and separately in the absence, of said ligand;
• wherein the concentration of said site-specific fluorescent probe is in the range of about 0.5 μM to about 20 μM, wherein said site-specific fluorescent probe:
• i) increases in fluorescence when bound to albumin, as measured by a spectral reading device;
• ii) binds specifically to a binding site I or a binding site II on said albumin;
• and
• iii) has a higher affinity for albumin in the denatured state than when said albumin is in its native state;
• and wherein ligand binding to binding site I or binding site II is measured by displacement of the site-specific fluorescent probe by said ligand when it binds to said albumin;
• b) measuring an initial level of fluorescence for the reaction mixture in the presence and separately in the absence of said ligand;
• c) comparing the initial level of fluorescence for the reaction mixture in the presence and the absence of said ligand;
• whereby differences in the level of the initial fluorescence in the presence or in the absence of said ligand indicates that said ligand does or does not, respectively, bind to said binding site I or binding site II on said albumin;
• d) heating said reaction mixture;
• e) measuring a change in fluorescence of the reaction mixture associated with the thermal unfolding of said albumin, resulting from said heating, in the presence and separately in the absence of said ligand;
• f) generating a thermal unfolding curve from the change of fluorescence measured in (e) for said albumin in the presence and separately in the absence of said ligand as a function of temperature for the reaction mixture;
• g) comparing said thermal unfolding curves for said albumin in the presence and the absence of said ligand generated in (f);
• whereby differences in the thermal unfolding curves are used to determine the affinity of the ligand which binds to albumin;
• and
• h) determining both the affinity and specificity of said ligand which binds to albumin by comparing the differences in the thermal unfolding curves in (g) and comparing the differences in the level of the initial fluorescence in (c);
• thereby determining both the affinity and specificity of said ligand which binds to albumin in a single assay.