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

V. Methodology
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molecule. The globulins are also globular proteins, but
many of them, in contrast to albumin, precipitate in pure
water and require salts to maintain their solubility. The
globulins are a mix of proteins of various types, which
migrate in groups in an electric field (electrophoresis) as
families of proteins identified as α -, β -, or γ -globulins. The
nomenclature of the globulin fractions is based on their
location during separation by electrophoresis. Albumin has
the most rapid migration of the major proteins (in some
species it is preceded by prealbumin), followed by the
α -globulin, β -globulin, and γ -globulin fractions, respectively.
The γ -globulins are largely composed of immunoglobulins,
the antibodies that bind to invading pathogens
or other foreign matter. In contrast, the α - and β -globulin
fractions contain a great variety of different proteins.
Electrophoresis is a well-established diagnostic method
that was first introduced to the clinical biochemistry laboratory
with cellulose acetate as the support medium for the
separation. This has largely been replaced with agarose, so
that serum protein electrophoresis (SPE) in agarose gels,
followed by protein staining and densitometry to quantify
the protein in each of the main fractions, is common
in clinical biochemistry laboratories. This has evolved
into an extremely useful technique because aberrations are
observed in many disease states though there are only a
few diseases where the electrophoretic pattern can provide
a definitive diagnosis.
Interest has advanced the investigation armory for
serum protein analysis with the development of specific
analytical methods for individual proteins. Though specific
assays have been used for a long time for determination
of proteins such as albumin and fibrinogen, it is only
relatively recently that specific assays for other diagnostically
useful proteins such as haptoglobin, CRP, SAA, and
α 1 -acid glycoprotein (AGP) have become commonly available.
In most cases, this has been achieved by the use of
immunoassays, which has often required the development
and validation of species-specific methodology.
V . METHODOLOGY
A . Total Protein
Assays for total protein can be performed on serum or
plasma. The method employed to measure the total amount
of protein in solution varies with the amount of dissolved
protein and is therefore chosen according to the biological
fluid under investigation. The technology used to measure
total protein can be based on chemical or physical measurements.
In the diagnostic laboratory, chemical methodologies
are used because they can be readily adapted
to automated analyses. On the other hand, point-of-care
determination of total protein, for instance, in a veterinary
practice, can be performed by use of refractometry, which
depends on the physical properties of protein in solution.
1 . Chemical Methods
a . Biuret Reaction
The biuret reaction, in which protein forms a complex with
copper (Cu 2 ) in alkaline solution, has become the standard
chemical test for total serum or plasma protein. This
complex, which is dependent on the presence of peptide
bonds, is blue-purple in color. This method is used in automated
wet biochemical analyzers and is also the basis for
total protein assays in dry chemistry analyzers. The biuret
method is highly accurate for the range of total protein
found in serum (1 to 10 g/dl, 10 to 100 g/liter) but is not
sensitive enough for the protein concentrations found in
other body fluids where the concentration range is lower,
for example, cerebrospinal fluid. More sensitive protein
assays should be used for these fluids.
b . Precipitation Methods
Proteins in solution are sensitive to changes in the pH of the
environment that result in alteration of the ionization of the
side groups of acidic and basic amino acids. This distorts
the electrostatic forces between residues, which normally
keep the protein in its native conformation. Changing the
pH, especially to the extremes of the pH range, therefore
disrupts the tertiary and quaternary structures of proteins
leading to reduced solubility and causing precipitation of
the protein from solution. Reagents such as trichloroacetic
acid, sulphosalicylic acid, and tungstic acid cause the
precipitation of protein and are used to quantify the total
protein concentration in biological fluids when the protein
concentration is in a range of 0.1 to 1g/dl (1 to10 g/liter).
c . Sensitive Chemical Methods
For measurement of total protein in fluids at concentrations
less than 0.1 g/dl (1 g/liter), more sensitive protein
assays have to be used. For many years, the Phenol-Folin-
Ciocalteau method ( Lowry et al. , 1951 ) was the method
of choice to measure low concentrations of protein. This
method is based on the reaction of the phenolic group of
tryptophan and tyrosine with the Folin-Ciocalteau reagent
yielding a blue chromogen. A less laborious modification of
this method, which is even more sensitive, has been developed
using bicinchonic acid ( Smith et al. , 1985 ), whereas
methods based on the binding of the dye, Coomassie blue
to protein in acidic solution, are also useful in quantifying
dilute protein solutions ( Bradford, 1976 ). These sensitive
methods are conveniently performed in microtiter plates,
but their use is mainly restricted to the research laboratory.
These sensitive methods depend on the reactions between
reagent and a number of specific amino acids in the proteins,
such as with the phenolic group of aromatic amino acids.
Results vary depending on the proportion of these amino
acids in the proteins being measured. The protein used to
calibrate the assay may have a significant effect on results.
Conventionally, bovine serum albumin is used as calibrant.