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We describe a double-antibody radioimmunoassay for the determination of free thyroxine (FT4) in serum using a 125I-labeled T4-IgG conjugate of very large molecular weight (approximately 1,000,000) as tracer. The binding of this conjugate to the anti-T4 antibody was found to be satisfactory and independent of the T4-binding protein concentration. The T4 sequestration by the antibody was limited and the sample dilution used in the final assay mixture was low. The results determined by the assay developed for samples from hypo-, hyper-, euthyroid subjects and from non-thyroidal-illness patients were well correlated with those obtained by a free T4-equilibrium dialysis kit (Nichols). The detection limit of the assay was 0.41 pmol/l and the range covered by assay standard solutions was 2.4-121 pmol/l. The FT4 concentrations in serum as determined by this assay were 9.6-31 pmol/l (mean 21 pmol/l) for 46 euthyroid serum samples; 6.7-47 pmol/l (mean 23.5 pmol/l) for 27 non-thyroidal illness; and 4.2-25.8 pmol/l (mean 11.6 pmol/l) for 19 pregnant women.

It is frequently difficult to establish a diagnosis of hyperthyroidism in association with pregnancy. Signs and symptoms suggestive of hyperthyroidism may occur in normal pregnancy. Moreover, tests to rule out hyperthyroidism under these conditions are not readily available. Mild hyperthyroidism is not a hazard to an otherwise normal pregnancy and does not require therapy on the basis of this presumption. Hyperthyroidism of a clinically significant degree is safely treatable by medical means without hazard to the fetus. Hyperthyroidism is an uncommon cause for the failure of pregnancy to proceed to term. Treatment with thyroid based on a presumption of this diagnosis is justified, but such patients should be studied carefully after delivery to establish the true state of thyroid function for the future. Other conditions of thyroid dysfunction, including thyroiditis, thyroid carcinoma, nontoxic goiter, and ovarian struma rarely interfere with an otherwise normal pregnancy.

A thyroxine radioimmunoassay procedure (T4RIA) based on incubation and separation on Sephadex columns is presented. The assay is rapid and easily to perform; if necessary columns may be regenerated. The raising of antibodies against thyroxine in goats is described in detail. The specificity of the antiserum towards the coupling of several compounds related to thyroxine has been tested. Influence of some buffers on the binding of thyroxine to serum proteins has been investigated. The results of T4RIA in patient sera were in agreement with those obtained by a competitive binding method. The within-day variation was approximately 4% (coefficient of variation, C.V.); day-to-day variation was 7% C.V.

A dual competitive protein-binding assay for serum thyroxine (T4) is described. The assay is called the "corrected" T4 index and can be performed simultaneously with the determination of total T4. This method correlated significantly with the free T4 index and the effective T4 ratio. The "corrected" T4 index was compared for precision and diagnostic accuracy with the effective T4 ratio and two different free T4 indices. Improved modifications of the thyroxine and triiodothyronine Sephadex uptake tests, used for calculation of the free T4 indices, are also reported. In spite of better precision, the "corrected" T4 index and effective T4 ratio gave twice as many incorect classifications of both hyper- and hypothyroidism as the free T4 indices. Previous reports on the effective T4 ratio and other basically similar tests might give an overly optimistic view of what can be expected in routine clinical practice. The free T4 index, which is a good estimate of the free T4 concentration, may still be the thyroid function test of choice when serum T4 and clinical impression are not in conformity.

We describe a double-antibody solid phase radioimmunoassay for free thyroxine (FT4) in serum with use of 125I-labelled thyroxine-human chorionic gonadotropin conjugate. Since the labelled conjugate does not bind to thyroxine binding globulin (TBG) and albumin because of its large molecular weight, the method is uninfluenced by TBG or albumin. The measurable range of FT4 in serum was 2.0 to 128 ng/l. The mean coefficients of variation within and between assays were 4.6-8.6% and 6.3-11.6%, respectively. The FT4 values determined by the proposed method correlated well with those determined by commercial radioimmunoassay in subjects with normal albumin concentration (r = 0.98). The FT4 concentrations in serum as determined by this method were 9 to 17 ng/l for healthy adult subjects; high for patients with hyperthyroidism; low for patients with hypothyroidism; and within normal limits for pregnant women, and patients with high or low concentrations of thyroxine-binding globulin.

In 28 patients with Graves' disease showing a wide range of thyroid function between the extremes of hypothyroidism and hyperthyroidism, the following parameters of peripheral thyroid function were measured: serum thyroxine concentration, serum-free thyroxine concentration, serum triiodothyronine concentration, and serum-free triiodothyronine concentration. In 25 patients, thyroxine turnover was also measured. Thyroxine turnover was found to be highly correlated with serum-free thyroxine concentration (r equals 0.9405) and serum-free triiodothyronine concentration (r equals 0.9184). Serum-free thyroxine fraction correlated with serum-free triiodothyronine fraction (r equals 0.8445), suggesting that similar factors in serum controlled the intensity of protein binding for both thyroxine and triiodothyronine. Thyroxine turnover calculated by a noncompartmental method agreed closely with values calculated by the compartmental method, suggesting that the former simpler method has general utility.

The structures of the carbohydrate moieties of three hormone-binding glycoproteins from human serum, namely, thyroxine-binding globulin, transcortin, and sex hormone-binding globulin, have been characterised using quantitative g.l.c. of the methylated monosaccharide derivatives obtained after methanolysis of the methylated glycoproteins.