Menetelmä vapaiden ligandien mittaamiseksi biologisissa nesteissä

The invention provides a method for measuring the concentration of free testosterone ligand in a biological fluid in the presence of bound ligand and endogenous binding proteins, without disturbing the equilibrium between free ligand and protein-bound ligand, which method comprises (a) incubating, in the absence of salicylate, 2,4-dinitrophenol and 8-anilino-1-naphthalenesulfonic acid, a sample of the biological fluid with (i) a ligand analog tracer which, due to its chemical structure, does not bind to some of the endogenous binding proteins but does bind to at least one other endogenous binding protein, (ii) a concentration of a specific ligand binder having an affinity constant and selectivity for the free ligand such that the equilibrium between free ligand and protein-bound ligand is not disturbed and (iii) a concentration of sulfobromophthalein (SBP) that inhibits the binding of the ligand analog tracer to said at least one other endogenous binding protein sufficient to block reaction between the ligand analog tracer and said at least one other endogenous binding protein without displacing ligand from protein-bound ligand; (b) separating the ligand analog tracer bound to the specific ligand binder from unbound tracer; and (c) determining the concentration of free ligand in said biological fluid.