The thyroid hormone, thyroxine (T4) is produced by the thyroid gland. An important component in the synthesis is iodine. The major form of thyroid hormone in the blood is thyroxine (T4). Thyroxine is converted to the active T3 (three to four times more potent than T4) within cells by deiodinases (5'-iodinase).

Thyroxine-binding globulin (TGB) is the major carrier protein for circulating thyroid hormone. Only a very small fraction of the circulating hormone is free (unbound) - T4 0.03%. When thyroid hormone is bound, it is not active, so the amount of free T3/T4 is what is important. For this reason, measuring total thyroxine in the blood can be misleading.

The concentration of free thyroid hormones in the blood is regulated by a negative feedback mechanism involving TSH. The binding of T4 by TBG plays a key role in this feedback mechanism and the most significant changes that occur in T4 binding capacity are the result of alterations in TBG. Changes in the circulating levels of TBG will result in a proportional increase or decrease in the concentration of total T4. However, measurement of serum free T4 is unaffected by changes in T4 protein binding levels and therefore correlates well with the functional thyroid state in most individuals. Factors responsible for discrepancies between serum total T4 levels and true thyroid states include TBG concentration, estrogenic hormones (pregnancy, oral contraceptives and estrogen) and drugs that bind to TBG preventing its binding to free T4.

The thyronines act on the body to increase the basal metabolic rate, affect protein synthesis and increase the body's sensitivity to catecholamines (such as adrenaline) by permissiveness. The thyroid hormones are essential to proper development and differentiation of all cells of the human body. These hormones also regulate protein, fat, and carbohydrate metabolism, affecting how human cells use energetic compounds. Numerous physiological and pathological stimuli influence thyroid hormone synthesis. Thyrotoxicosis or hyperthyroidism is the clinical syndrome caused by an excess of circulating free thyroxine, free triiodothyronine, or both. Both T3 and T4 are used to treat thyroid hormone deficiency (hypothyroidism).

May be diagnosed by:

• Determination of specific antibodies based on the ELISA technique
• Determination of specific antibodies based on the RIA-technique

Free T4 ELISA:

Competitive immunoenzymatic colorimetric method for quantitative determination of free T4 (Thyroxine) in serum or plasma.

Principle of the Assay:

Microtiter strip wells are precoated with anti-T4 antibodies (solid-phase). Free T4 in the sample competes with added horseradish peroxidase labelled T4 (enzyme-labelled antigen) for antibody binding. After incubation a bound/free separation is performed by solid-phase washing. The immune complex formed by enzyme-labelled antigen is visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product. The intensity of this product is inversely proportional to the amount of free T4 in the sample. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorption at 450 nm is read using an ELISA microwell plate reader.

Specific performance characteristics: 

Correlation with RIA performed on 59 samples is r = 0.978

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