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

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Chapter | 21 Clinical Reproductive Endocrinology
to prepare derivatives containing active groups such as carboxyl
or amino groups. These groups are then activated so
that they react with amino or carboxyl groups of the protein
molecule. The specificity of the antisera obtained by
immunization with a steroid-protein conjugate depends
on the site used for conjugating the steroid to the protein.
More specific antisera are obtained if the hapten (steroid) is
attached to the protein at a site remote from the characteristic
functional groups of the hormone (Lindner et al. , 1972).
The species most frequently used to produce polyclonal
antibodies are sheep and rabbits. One of the most popular
and efficient schedules for immunization involves multiple
injections in the back and neck of the animal of the antigen
emulsified in complete Freund’s adjuvant (Vaitakaitus
et al. , 1971). During immunization, the developing antibody
titer is monitored, and a relatively large number of milliliters
of serum can be obtained when a suitable titer has been
achieved. A few milliliters of a high titer antiserum are usually
sufficient for millions of immunoassay determinations.
Antisera seem to be stable when stored at 20 °C, although
the usual preferred temperature is 70 °C.
The discovery that hybridomas could be used to produce
an endless supply of antibodies with certain specificity
( Köhler and Milstein, 1975 ) led to a new development
in the technology of antibody production. The procedure
involves the fusion of two cell lines: B lymphocytes selected
for the production of a specific antibody and myeloma cells
that have the capacity for permanent growth. Antibody
production occurs by injection of the cell lines into mice;
permanency is assured by maintaining a supply of cells in
the frozen state. Monoclonal antibodies are useful for the
quantitative immunoassay of hormones. They have the
advantages of specificity, unlimited supply over time, and
the possibility to standardize assay methods between laboratories;
disadvantages are that they have lower affinities as
compared to polyclonal antibodies and they do not always
form precipitates with antigens. Mixing of monoclonal antibodies
may increase affinity ( Ehrlich et al. , 1982 ).
2 . Labeled Hormone
In radioimmunoassay techniques for polypeptide hormones,
the antigen (hormone) is most commonly used
for preparing the radioactive tracer. Usually, radioactive
iodine, 125 I, is used for radioiodination of the antigen. The
two most frequently used techniques for iodination are the
chloramine-T ( Hunter and Greenwood, 1962 ), immobilized
chloramine-T (Iodobeads; Markwell, 1982 ), and the
lactoperoxidase procedures ( Thorell and Johansson, 1971 ).
Peptide hormones containing tyrosyl or histidyl residues
can also be iodinated with these techniques.
Many RIA systems for steroid hormones and prostaglandins
utilize tritiated forms of these molecules, which
are available commercially. Because tritium has a considerably
longer half-life than iodine, tritium tracers can be
used in many cases over several years, whereas the iodinated
tracers often have to be prepared monthly. There are,
however, certain advantages in using iodinated tracers for
steroid hormones and prostaglandins in that simpler and
cheaper counting systems can be used (i.e., gamma counting
as opposed to liquid scintillation counting). Another
advantage of radioiodine over tritium is its higher specific
activity, which increases the sensitivity of the assay.
Direct incorporation of iodine in the skeleton of steroid
hormones results in a loss of the immunoreactivity. Thus,
the approach taken for radioiodination of steroid hormones
has been to link a tyrosyl or histidyl molecule to the steroid
molecule, making direct radioiodination possible
( Niswender, 1973 ), or to iodinate a compound such as tyramine
and conjugate the iodinated compound to the steroid
molecule ( Lindberg and Edqvist, 1974 ).
Most EIA systems for steroid hormones use horseradish
peroxidase, alkaline phosphatase, or β-galactosidase as
labels. The enzyme-labeled steroid is produced the same
way as has been described for the synthesis of steroid protein
conjugates for the production of antibodies.
3 . Separation of Antibody-Bound and
Free Hormone
An essential part of any immunoassay system is an efficient
procedure for the separation of antibody-bound and
free hormone. Several different approaches have been taken
to achieve a rapid and efficient separation. Frequently used
separation procedures are based on antibodies coupled to
an insoluble polymer, precipitation of antibodybound hormone,
or adsorption of free hormone.
Antibodies coupled to an insoluble polymer can be used
for separating antibody-bound and free hormone in RIA
procedures ( Abraham, 1969 ). One procedure involves the
decanting of polystyrene tubes in which antibodies have
been adsorbed to the surface of the tube, which is followed
by determination of radioactivity in the antibody-bound
(contained in the tube) or free (contained in the eluent)
form. In most EIA systems designed for low-molecularweight
hormones, antibody-bound hormone is measured
after free hormone has been removed by washing the wells
of the microtiter plate that have been previously coated
with antibody.
Another common procedure for RIA utilizes antibodies
covalently coupled to an insoluble polymer granule. In
this case, free and antibody-bound hormones are separated
through centrifugation. After removal of the supernatant
containing the free hormone, the antibody-bound radioactivity
can be determined. Antibody-coated glass beads have
been used in both RIA and EIA ( Schmidt et al. , 1993 ).
Separation is achieved by washing the bead and then the
radio or enzyme activity is determined.
Precipitation of the antibody-bound hormone has been
achieved through the addition of ammonium sulfate ( Mayes