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

II. Assay Methods
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compounds), need for well-trained personnel and specialized
laboratory, and problems in disposal of radioactive waste.
Consequently, great progress has been made to develop assays
that use nonisotopic labels such as enzymes, fluorogens, and
chemiluminescent precursors. Enzyme immunoassays can be
as sensitive, accurate, and precise as radioimmunoassays; the
specificity depends on the quality of the antibody as is true for
radioimmunoassay systems ( Munro and Stabenfeldt, 1984 ).
Of particular interest in domestic animals has been their use
in the determination of progesterone in blood ( Lopate and
Threlfall, 1991 ; Meyers et al. , 1988 ; Munro and Stabenfeldt,
1984 ) and in milk ( Allen and Foote, 1988 ; Arnstadt and
Cleere, 1981 ; Etherington et al. , 1991 ; Sauer et al. , 1981 ).
Enzyme immunoassay in the laboratory setting is very efficient,
particularly as concerns the time required for the assay
reaction (2 h or less), ease of separation of bound from free
hormone when using microtiter plate system (30 sec to wash 96
wells on a microtiter plate), and speed of end point analysis
(optical densities can be determined in 1 min for a 96 well
microtiter plate).
Among the various approaches to enzyme immunoassay,
the double antibody sandwich method for determination
of larger hormones is frequently used. In this
assay system, the plastic wells are coated with antibody.
The sample to be processed is then added, and its hormone
binds to the antibody-coated plastic well. A second
enzyme-labeled antibody directed against another epitope
of the hormone is then added. The amount of enzymelabeled
antibody bound is directly proportional to the
amount of hormone in the sample. There are two advantages
with this methodology: (1) the hormone does not
need to be isolated for labeling, and (2) the same general
technique can be used to label different antibodies. The
method can only be used to assay hormones with at least
two binding sites, and it is thus unsuitable for measurement
of low-molecular-weight hormones like steroid hormones.
The analytical equipment used for enzyme immunoassay
can be used for a variety of determinations including
those involved in disease surveillance and drug analysis.
This allows for the sharing of one specialized spectrophotometer
among several disciplines at a great reduction
in cost. Another benefit is that the analytical equipment
needs little maintenance. The elimination of the problems
engendered by use of radioisotopes is a major advantage
for enzyme immunoassay. This and the fact that color
change is fundamental to enzyme immunoassay means that
the assay can be used visually to determine the presence
or absence of a corpus luteum (CL) in domestic and other
species. Enzyme immunoassay-based analytical systems
for progesterone in blood or milk can be used on the farm
beside the animal to directly assess the functional ovarian
status of an animal ( Herrler et al. , 1990 ; Matsas et al. ,
1992 ; Nebel et al. , 1989 ; Romagnolo and Nebel, 1991 ).
Automatized immunoassay instrumentation has been
developed mainly for the human medical market, but some
systems have specific veterinary applications (e.g., chemiluminescent-labeled
immunoassays by DPC) , and as some
hormones (e.g., steroids) cross-react between species, these
automatized immunoassay instruments can be useful in a
veterinary diagnostic lab with high throughput.
A . Immunoassay
1 . Production of Antibodies
Immunoassay techniques utilize antibodies as binding
proteins. Hormones such as LH and FSH, which are glycoproteins
with molecular weights of around 30,000, are
antigenic because of their size and chemical composition.
In general, a lower level of purity is required of the polypeptide
hormones for antibody production as compared to
the hormone used in the labeling procedure.
If an assay for bovine LH utilizes an antibody to bovine
LH, radio- or enzyme-labeled bovine LH as tracer, and
bovine LH as the standard, the assay system is completely
species specific and is said to be of the homologous type.
Such a system represents the ideal immunoassay system
for measuring a polypeptide hormone. Because of the limited
availability or lack of suitable purity of polypeptide
hormone preparations, heterologous assay systems have
been developed. In these cases, an antiserum to a polypeptide
hormone of one species has been used to determine the
same polypeptide hormone in another species. The standard
hormone used for quantification of the assay should,
however, originate from the same species for which the
measurements are performed.
Polypeptide hormone antisera are available that show
a high degree of cross-reactivity. One such antiserum of
special interest in the field of reproductive hormones in
domestic species is a polyclonal LH antiserum raised
against ovine LH ( Niswender et al. , 1969 ). This antiserum
reacts specifically with LH from other species and has been
used to determine LH in approximately 45 species including
the cow, sheep, pig, cat, and dog ( Madej and Linde-
Forsberg, 1991 ; Millar and Aehnelt, 1977 ). A monoclonal
antibody generated against bovine LH has been reported to
have high cross-reactivity between species ( Bravo et al. ,
1992 ; Forsberg et al. , 1993b ; Matteri et al. , 1987 ).
Steroid hormones and prostaglandins have considerably
lower molecular weights and thus are not immunogenic
per se. However, these structures can be rendered immunogenic
if covalently linked to large carrier molecules such as
bovine serum albumin, and specific antibodies can be elicited.
For such a hormone-protein conjugate to be immunogenic,
approximately 10 to 20 hormone molecules should
be present per molecule of protein. In the case of bovine
serum albumin, about 30% of the sites available for conjugation
should be occupied.
Most naturally occurring steroid hormones and prostaglandins
contain hydroxyl or ketone groups, which are used