Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

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Chapter | 21 Clinical Reproductive Endocrinology
techniques for the quantitative determination of immunoglobulin
G ( Engvall and Perlmann, 1971 ). The techniques
are competitive and utilize the same basic principle,
which is based on the ability of nonlabeled hormone to
compete with a fixed amount of labeled (radioactive isotope
or enzyme) hormone for the binding sites on a fixed
amount of protein. The nonlabeled hormone reduces the
number of free binding sites on the protein, thus decreasing
the availability of the binding sites to the labeled hormone.
At equilibrium, the free hormone is separated from
the protein-bound hormone, and the reaction is quantified
by the determination of the amount of labeled hormone
that is antibody bound or free ( Fig. 21-3 ). The degree of
inhibition of binding of the labeled hormone to the binding
protein is a function of the concentration of nonlabeled
hormone present in the solution. As a basis for the quantification,
a standard curve is developed with fixed amounts
of labeled hormone and binding protein incubated together
in the presence of a known and graded concentration of
unlabelled hormone ( Fig. 21-3 ).
Certain disadvantages exist to the use of radioisotopes as
labels in immunoassays. Among those are limited shelf life
and stability of radiolabel compounds, need for relatively
expensive counting equipment (especially for tritium-labeled
Binding
protein
Radioactive
hormone
Non radioactive
hormone
% Radioactive
hormone bound
to binding protein
Free
hormone
0 pmol
100%
2 pmol
66%
4 pmol
50%
8 pmol
33%
% radioactivity bound
100
75
50
25
0
2 4 8
% radioactivity bound
100
75
50
25
0
2 4 8
Concentration of hormone pmol
logit
2
1
0
1
2
2 4 8
FIGURE 21-3 The principle of an immunoassay technique is based on the ability of the binding protein to bind the labeled hormone. Excess-labeled
hormone as added to ensure saturation of the hormone-binding sites on the binding protein. The addition of increasing amounts of nonlabeled hormone
(2,4, and 8 pmol) results in a proportional decrease in the quantity of labeled hormone bound to the binding protein. Separation of labeled hormone
bound to the binding protein from free hormone must be achieved before quantification can be done. In the lower part of the figure, this reaction is
depicted in three different ways. In the panel to the left and in the middle, the percentage-labeled hormone bound to the binding protein is on the ordinate,
and the amount of hormone on a linear scale (left) and log scale (middle) is on the abscissa. In the panel to the right, the logit of the response
variable is on the ordinate, and the amount of hormone is plotted on a log scale. The method depicted in the middle and to the right can be used for
determination of parallelism. The logit/log transformation (right) is frequently used when computers analyze immunoassay data.