Abstract: A biological sample containing an analyte, partly free and partly bound to natural binders, is incubated with a labelled specific binder for the analyte and a derivative of the analyte which does not react with the natural binders. Part of the labelled specific binder binds to the analyte derivative, the proportion depending on the free analyte concentration in the sample. The proportion is measured and used to determine the free analyte concentration. For example, the analyte may be thyroxine or cortisol, the labelled specific binder may be an antibody thereto, and the analyte derivative may be in the form of a solid matrix. The labelled specific binder should have an affinity for the analyte, expressed as a dissociation constant of the complex of the two, approximately equal to the concentration of free analyte in the sample.

Abstract: Novel amides of an iodothyronine compound such as tri-iodothyronine (T3) or thyroxine (T4) with an aminoacetic acid compound such as nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA) or diethylenetriamine pentaacetic acid (DTPA) may be labelled, e.g. radioactively labelled with I-125, to give labelled derivatives useful in assays of a free thyroid hormone in a biological fluid which also contains the thyroid hormone bound to one or more natural binders. The novel compound T4 EDTA is shown to bind much more strongly to antibodies to T4 than to the natural protein binders in the biological fluid.

Abstract: A method of determining the concentration of the free portion of a ligand present in a biological fluid which also contains the ligand bound to one or more natural binders involves the single step of mixing a sample of the fluid with a labelled derivative of the ligand and a specific binder for the ligand, incubating the mixture, and determining the proportion of the labelled derivative of the ligand bound to the specific binder. The labelled derivative of the ligand is chosen to bind strongly to the added specific binder, but to bind not at all, or much more weakly than does the ligand, to the natural binders in the biological fluid. A small amount of the labelled derivative can be used, and the binding equilibrium of the ligand is not appreciably perturbed. Ligands include thyroxine, tri-iodothyroxine and cortisol.