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LABORATORY MEASUREMENT OF HORMONES

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Estimation of a hormone level in terms of STRUCTURAL MASS can be performed by
immunoassay or immunometric assay using radioactive, fluorescent, luminescent or
enzyme-linked markers
Also performed by receptor assay (ligand binding assay)
Estimation of a hormone level in terms of ACTIVITY is performed using a bioassay
All systems calibrate the hormone level in the unknown sample against a series of
defined “standards” on the basis of a response comparison in the test assay system
A comparison of the response produced by the hormone in the sample, and the
responses produced by different known concentrations of a reference preparation of
the same hormone

IMMUNOASSAYS
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2 Types namely immunoassay proper of which we use radioimmunoassay as an
example, and immunometric assay of which we use immunoradiometric assay as an
example
There is a specific interaction between the antibody and the antigen, in this case the
hormone
Hormone and a specific antibody form an antibody-hormone complex
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Hormones to which unknown hormonal levels in blood sample are calibrated against can be
labelled in the following ways:
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Radioactive(e
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e
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If the hormone level in a blood sample is low, then a higher level of labelled (e
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radioactive) hormone will be bound to the antibody (i
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remaining in the incubation
tube) following removal of unbound hormones [hormones (both labelled and
unlabelled) that were not bound to the antibody as there is only a finite amount of
antibodies]

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Xd represents the concentration of unlabelled hormone and xr represents the
intensity of the signal generated by tagged hormones, i
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radioactivity
Increasing the concentration of unlabelled hormone decreases e
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the level of
radioactivity measured (low intensity) because the unlabelled hormone
outcompetes the labelled hormone for antibody binding
The curve generated is sigmoidal

IMMUNOMETRICASSAY: IMMUNORADIOMETRIC ASSAY:
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These use two antibodies (e
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monoclonals antibodies which bind to different
regions (epitopes) of a single hormone molecule
One of the antibodies is bound to the surface of the test tube (side) and also to a
hormone epitope
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When it binds the hormone in the blood sample (unknown levels), it
generates a signal (radioactivity)
The higher the levels of hormone in the blood sample the higher the radioactivity
detected
This form of immunoassay is also called sandwich assay

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Linear response
Increase in hormone concentration increases intensity of signal (radioactivity)
because hormone number 2 binds to the epitope of each hormone

ALL IMMUNOASSAYS ARE:
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Sensitive (can measure differences in levels);
Specific (can distinguish between similar structures, e
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LH, FSH and TSH);
Precise (give high degree of confidence that the measured value is repeatable);
Convenient (cost-effective, kit-based, ease of operation, high sample throughput)
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The hormone however may not bind its target receptor on the cell
Immunoassays may also present results implying low levels of a particular
hormone in blood when in reality there are correct levels of a particular hormone in

blood but the problem lies in an impaired antigenic site on the hormone where the
antibody binds, yet the hormone is still able to bind to the target cell via a different
epitope and brings about a cellular response
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Increase in hormone concentration increases a particular response
Sigmoidal

Example:

Bioassays: limitations and advantages:
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In vivo bioassays: - laborious; technically-demanding; expensive; insensitive;
poorly-reproducible; subject to species-specificity limitations
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ALTERNATIVE TO IMMUNOASSAYS
Receptor assay / Ligand binding assay

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Competition between labelled hormone and hormone in blood sample (unknown
levels)
They both compete for a finite amount of receptors
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g
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e
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