Abstract: Disclosed is a method for the direct determination of the free fraction of analytes present in biological fluids in a free form and in a form bound to one or more endogenous ligands (said free and bound forms being in equilibrium with one another). This method provides for a (preferably substantially simultaneous) use:a first ligand L1 capable of sequestering an analyte quantity proportionate to the free-analyte concentration present in a biological fluid and to subsequently release it, after removal from the biological fluid of the specific endogenous ligand, as a result of the addition of an appropriate selective dissociating agent; a second ligand capable of binding both the previously released analyte and a labelled version of the analyte, even in the presence of the dissociating agent; a selective dissociating agent; and a quantity of labelled analyte.

Abstract: A method for direct determination of the free fraction of an hormone in a biological fluid containing bound and unbound hormone in an equilibrium state. A fluid sample is contacted with a solid matrix capable of reversibly binding the free hormone without sensibly affecting the equilibrium state and the amount of adsorbed hormone is determined and directly correlated with the free hormone in the biological fluid. The method is particularly suitable for direct determination of free thyroxine and triiodothyronine in serum.