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Nussey S, Whitehead S. Endocrinology: An Integrated Approach. Oxford: BIOS Scientific Publishers; 2001.

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Endocrinology: An Integrated Approach.

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Box 3.30Measurement of ‘free’ thyroxine — indirect binding assay

Image ch3fb30.jpg

Image dclcc1.jpg Free T4 in patient's serum is captured on antibodies, and hormones bound to plasma globulins are removed. Latex particles are recovered by binding to an inert glass fiber matrix.

Image dclcc2.jpg Latex particles are incubated with T4 labelled with alkaline phosphatase. The T4 binds to any unoccupied antibody binding sites on the latex particles. Excess T4 is removed.

Image dclcc3.jpg Latex particles are incubated with 4-methylumberlliferyl phosphate which fluoresces when its phosphate group is removed by the alkaline phosphatase on the T4.

Image dclcc4.jpg The intensity of fluorescence is indirectly proportional to the concentration of free hormone in the serum sample and comparison of the results with those from tubes containing known amount of T4 allows calculation of the original concentration of free hormone.


  • There is no international standard for T4 or T3 assays.
  • The direct measurement of free T4 and T3 requires physical removal of bound hormones by dialysis or unfiltration. Such assays are expensive and labor intensive and, though used for research studies, are not suitable for routine laboratory use.
  • Total serum concentrations of T3 and T4 are measured by direct competitive binding assays.

From: Chapter 3, The thyroid gland

Copyright © 2001, BIOS Scientific Publishers Limited.


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