Chemistry: analytical and immunological testing – Thyroid hormone tests
Patent
1985-02-20
1988-05-17
Marantz, Sidney
Chemistry: analytical and immunological testing
Thyroid hormone tests
436501, 436534, 436545, 436804, 436817, G01N 3353, G01N 33534, G01N 33546, G01N 3378
Patent
active
047450720
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method for measuring the concentration of free ligand in a biological fluid containing free ligand and ligand bound to endogenous binding agent.
BACKGROUND ART
Immunoassay techniques have been developed in recent vears to measure concentrations of free hormones and other ligands in sera and other biological fluids which contain free ligand in equilibrium with ligand bound to endogenous binding agents such as binding proteins. They are based on the principle that if a specific binder for the ligand, usually an antibody, is brought into contact with the sample to be tested the extent of occupancy of the binding sites on the specific binder by the ligand is a measure of the concentration of free ligand, provided that the amount of specific binder is sufficiently low that the equilibrium between free and endogenously bound ligand is not significantly affected. By measuring the extent of occupancy for the unknown sample and calibrating such a measurement using standard samples containing known free ligand concentrations it is possible to determine the free ligand concentration in the unknown sample.
Initially, the extent of occupancy of binding sites was measured by removing the specific binder containing bound ligand from the sample and determing the proportion of unoccupied sites by back-titration using an appropriately labelled material (e.g. radioactively labelled material) which binds at the unoccupied sites. The process was thus effectively a two-step process.
Subsequently it has been proposed to carry out the `back-titration` without removing the specific binder from the sample, thus converting the two-step process into a one-step process. This can be done either by using as the labelled material a labelled analogue of the ligand or by using as the labelled material a specific binding agent.
Thus, it has been proposed in published European Patent Application No. 0,026,103 which is equivalent to U.S. Pat. No. 4,366,143 of Midgley et al to measure the concentration of free ligand in such a biological fluid by a radioimmunoassay technique comprising (a) admixing a sample of the fluid with a labelled derivative of the ligand and with a specific binder for the ligand, (b) effecting reaction between the free ligand, the labelled derivative and the specific binder, (c) if necessary, separating that portion of the ligand and labelled derivative that has become bound to the specific binder from that portion not so bound, (d) measuring the amount of the labelled derivative that is, or is not, bound to the specific binder, and (e) using that measurement to determine the concentration of free ligand in the biological fluid. According to the process disclosed there, 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 endogenous binding agent.
In an alternative procedure a method of determining the free ligand concentration involves an immunoradiometric assay comprising admixing a sample of the fluid with a labelled specific binder and an unlabelled analogue of the ligand, incubating the resulting mixture to permit the free ligand and the unlabelled analogue to compete for the labelled specific binder, determining the amount of labelled specific binder bound either to the ligand or to the unlabelled ligand analogue, and correlating the amount of bound labelled specific binder to the amount of free ligand present in the sample.
However when practical assay kits embodying the principles of EPA No. 0,026,103 have been employed to assay free thyroid hormone in samples taken from patients suffering, for example, from certain non-thyroidal illnesses or having serum protein abnormalities unrelated to free thyroid hormone concentration, the assay results appear to show an anomalous free thyroid hormone concentration, contrary to the correct position. It has also been found that the concentration of antibody (acting as specific binder) in those kits can be up
REFERENCES:
patent: 4366143 (1982-12-01), Midgley et al.
patent: 4376110 (1983-03-01), David et al.
patent: 4381291 (1983-04-01), Ekins
patent: 4522922 (1985-06-01), Carro
patent: 4536479 (1985-08-01), Vander-Mallie
Free Hormones in Blood: The Concept and the Measurement, Roger P. Ekins, Journal of Clinical Immonoassay, Summer 1984, pp. 163-180.
Ekins Roger P.
Jackson Thomas M.
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