Abstract: A one-step assay for the free portion of a ligand in a biological sample involves incubating a mixture of the sample with a labelled antibody for the ligand and a ligand analogue which competes with the ligand for binding to the antibody. The assay is characterized by choosing a ligand analogue which has a lower affinity for the antibody than does the ligand. An insolubilised ligand analogue preferably has a binding affinity for the antibody from 0.01% to 10% of that of the ligand. Ligand/ligand analogue pairs exemplified are T4/T3 (Thyroxine/Tri- iodothyronine); Testosterone/etiocholanol; and T3/T2.

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: 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.