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

V. Methodology
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has occurred will show an increase in turbidity, whereas a
sample from a neonatal animal that has not absorbed colostral
antibody will remain clear. The simplicity of these
methods has allowed their use as point-of-care methods.
A recent reevaluation of the use of sodium sulphite found
that a concentration of 18% (w/v) provided the optimal
diagnostic value. With zinc sulphate as the precipitant,
a similar diagnostic value was found, but it was affected
by hemolysis and the test solution was not stable when
exposed to atmospheric carbon dioxide ( Tyler et al. , 1999 ).
An alternative approach to assessment of antibody
transfer, which also causes the precipitation of the
γ -globulin fraction, is the glutaraldehyde coagulation test
( Sandholm and Kivisto, 1975 ). Glutaraldehyde reacts with
free amino groups on proteins causing cross-links to form
between protein chains and, if sufficient numbers are produced,
will cause aggregation of the proteins and visible
precipitate formation. γ -Globulins have the highest proportion
of the basic amino acids (lysine, arginine), which
have free amino groups available for reaction with glutaraldehyde.
Therefore when mixed with glutaraldehyde at a
predetermined concentration, a serum sample with a high
γ -globulin concentration will form cross-links and precipitate
formation will be visible, whereas a sample with low
γ -globulin will not produce a precipitate. However, fibrinogen
can also form cross-links with glutaraldehyde ( Liberg
et al. , 1975 ) and can cause interference with the test when
it is used for antibody detection in plasma, especially in
cases of hyperfibrinogenemia. Indeed, though the glutaraldehyde
test was deemed to have poor sensitivity and specificity
for detection of failure of passive transfer in calves
( Tyler et al. , 1996 ), it was found to be a useful screening
test able to distinguish between acute and chronic disease
in horses ( Brink et al. , 2005 ) because of this reaction with
fibrinogen in plasma.
The seromucoid fraction of serum is a group of highly
soluble glycoproteins that have the ability to remain in
solution in the presence of perchloric acid while other
proteins are precipitated. Methods have been developed
to estimate this “ acid soluble glycoprotein ” fraction by
addition of perchloric acid to aliquots of serum ( Nagahata
et al. , 1989 ). As most of the glycoproteins in the seromucoid
(acid soluble glycoprotein) fraction are APP, this was
an early means for monitoring the acute phase reaction.
2 . Dye Binding and the Albumin:Globulin Ratio
Albumin has the highest concentration of any of the individual
serum proteins, and valuable diagnostic information
can be obtained by measurement of its concentration.
Various dyes have been found that, after binding to albumin,
change their absorbance and are used as a means to
measure the protein in a spectrophotometer, in automatic
analyzers, or in dry chemistry systems. The dyes used most
widely for this procedure are bromocresol green (BCG)
and bromocresol purple (BCP). BCG has become the
favored dye to utilize in dye-binding assays for albumin
( Keay and Doxey, 1983 ), although BCP has been recommended
for equine serum albumin ( Blackmore and Henley,
1983 ). Accuracy of the dye-binding methods is generally
acceptable within the albumin concentration reference
ranges found in animals. However, the accuracy decreases
outside the reference ranges and may be unacceptable at
very low or very high albumin concentrations. With heparinized
canine plasma, it is possible that interference from
fibrinogen can lead to overestimation of the albumin concentration
when using BCG ( Stokol et al. , 2001 ).
Once the albumin and total protein concentrations have
been determined, the globulin fraction can be calculated by
subtraction of the albumin from the total protein concentrations.
The albumin:globulin ratio can then be calculated (albumin
concentration/globulin concentration). This provides a
means of assessing the relative contribution of the albumin or
the globulins to the total serum protein, which complements
the analysis of either analyte alone (Section VII.B) .
C . Electrophoretic Fractionation of the
Serum Proteins
Electrophoresis is the method of choice for analytical separation
of protein. Serum protein electrophoresis (SPE) is
currently regarded as the standard of reference for fractionation
of serum protein. Serum rather than plasma is
used as the sample for electrophoretic separation because
it reduces the complexity of interpretation by the removal
of fibrinogen. Many modifications have been made to the
basic principles of electrophoresis since the separation of
protein in an electric field was first pioneered by Tiselius
( Tiselius, 1937 ). Many of these methods have been applied
to serum proteins, but only a few have been employed in
clinical biochemistry. A major difference between methods
is the nature of the support material for the protein separation.
From the mid-20 th century, the cellulose acetate
membrane was utilized for this purpose for SPE ( Kohn,
1957 ). Toward the end of the century, electrophoresis on
agarose gel was introduced and has become more popular
in diagnostic laboratories. In contrast, biochemical research
laboratories almost universally use polyacrylamide gel as
the separation medium. Major advances have been made
in the ability to separate protein with the introduction of
two-dimensional electrophoresis (2DE) and associated
proteomic techniques. Whereas SPE on agarose can separate
serum into seven or eight fractions, it is claimed that
proteomic methodologies can separate and also identify
several hundred proteins simultaneously. Although these
new methods have not been validated for use in domestic
animal clinical biochemistry, it is valuable to be aware of
the possibilities that could be available by application of
these methods.