Capture Reagent and Strategy for Retrieving Albumin-Bound Ligands from Plasma

A capture strategy is described and demonstrated for retrieving ligand entities in plasma that bind Human Serum Albumin. The method has applications for both exogenous and endogenous ligands. Exogenous ligands include drug candidates, performance enhancing drugs and toxic nerve agents that also interact quite strongly with HSA. Endogenous ligands are natural circulating compounds whose abundance corresponds to normal hemostasis or elevated levels that could be disease-specific molecular biomarkers. Melting curves of plasma solutions measured by differential scanning calorimetry produce “so-called” plasma thermograms that are physical signatures of the plasma solution. Patterns displayed by thermograms can be sensitive indicators of the presence of abnormal levels of exogenous and endogenous ligand components. Effects of ligand interactions on thermodynamic stability of proteins in plasma that they bind, primarily HSA, manifest on the plasma thermogram. The capture strategy is demonstrated for HSA binding in plasma of four “ideal” ligands of different types. The particular ligands were naproxen, bromocresol green, short double stranded and single strand DNA. Thermogram shapes and features were sensitive to the presence of ligands as thermograms of mixtures of plasma and HSA with these ligands were significantly different than thermograms of plasma or HSA alone. These results demonstrated directly that significant perturbations of plasma thermograms corresponded to ligand interactions with HSA in plasma.