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

V. Methodology
131
D . Specific Protein Analysis
Serum protein is composed of many different individual
proteins with current electrophoretic fractionation in routine
diagnostic use (SPE) only providing a guide to the diseaserelated
changes affecting serum proteins as only a small
number of protein fractions can be consistently characterized.
Undoubtedly the ideal diagnostic approach would be to
monitor the changes in concentration of most if not all serum
proteins, and this is the ultimate objective of proteomic
investigation. However, until the technology advances sufficiently,
the only means to measure changes in individual
serum proteins is to use assays that directly measure the specific
proteins. In recent years, there has been increasing success
in identifying proteins with sufficient diagnostic value
to develop suitable assays to perform routine analysis. Some
serum proteins can be measured by methods where physical,
chemical, or biological activities can be exploited (Sections
V.A.2.b and V.B.2). However, most serum proteins are measured
by immunoassay requiring a specific antibody raised
against the target serum protein. Though there is cross reactivity
between species for a number of serum proteins, it is
advisable to use species-specific antiserum or to thoroughly
validate assays developed with antisera to species other than
the one under investigation.
1 . Immunoassays for Serum Proteins
Immunoassays have become an established weapon in the
arsenal of the clinical biochemistry laboratory, especially
where the exquisite specificity of antibody can be harnessed
for diagnostic procedures. Antibodies for use in immunoassays
for serum proteins can be polyclonal or monoclonal.
They are usually raised against the proteins purified from
serum, though a recombinant protein may be produced if the
gene sequence is known. There are several ways in which
antibodies can be incorporated in immunoassays to provide
qualitative or quantitative data with the choice of method
being dependent on several factors. The range of analyte concentration,
time taken to run an assay, and ease of automation
are among the considerations taken into account when
setting up an immunoassay for a specific serum protein.
a . Radial Immunodiffusion
One of the simplest methods for measurement of specific
serum protein is radial immunodiffusion (RID). This
method requires polyclonal antibody. The method is based
on the precipitation in agarose gel of antigen-antibody complexes,
and this does not occur with monoclonal antibodies
as more than one binding site on the antigen is required for
complex formation. The RID plates are prepared with agarose
gel containing antibodies to the protein antigen at an
optimized concentration. Sample is placed in a well in the
agarose and allowed to diffuse for 24 to 48 hours. A precipitin
ring forms because of the antibody-antigen reaction, the
diameter of which is dependent on the analyte concentration.
The concentration of the protein in the sample is determined
by comparison to standards. Radial immunodiffusion
assays have been used to measure immunoglobulin and
complement in serum and can distinguish between the different
classes of antibody (see Chapter 6) , and RID assays
for acute phase proteins have also been developed ( Ohwada
and Tamura, 1995 ; Tamura et al. , 1989 ).
b . Immunoturbidimetry
Immunoturbidimetry (IT) and the related method of immunonephelometry
also make use of the formation of antigenantibody
complexes, but in solution rather than in agarose
gel. With the correct balance of antigen and antibody, the
formation of antigen-antibody complexes can be followed
in a spectrophotometer as flocculation occurs and absorbance
increases. As the reaction takes as little as a few
minutes, this is the method of choice for automation of
analysis, but it is only suitable for protein concentrations
above 0.5 to 1.0 mg/dl (5 to 10 mg/liter). This method is
widely used in human clinical biochemistry for determination
of protein such as CRP, but availability of suitable
reagents has held back applications in veterinary medicine.
However, IT methods for canine CRP ( Eckersall et
al. , 1991 ) and feline AGP ( Bence et al. , 2005 ) have been
described. Although commercial kits for human CRP based
on IT have been validated for use in serum from some animal
species ( Kjelgaard-Hansen et al. , 2003 ), care has to
be taken in their use, especially as antiserum batches may
have differing cross-reactivities with animal protein so that
batch-to-batch variation may occur. Immunonephelometry
is a related method where reflected rather than absorbed
light is measured, which aids in reducing interference.
Another modification of the IT test is to make use of antibody-coated
latex particles, which can make assays more
sensitive as well as stabilizing the antibodies. A method
using latex particles coated with antibody to human serum
amyloid A has been validated to detect this protein in
horses ( Jacobsen et al., 2005a ; Stoneham et al. , 2001 ).
c . Enzyme-, Luminescent-, and Fluorescent-Linked
Immunosorbent Assays
Enzyme-linked immunosorbent assay (ELISA) are a common
format for many immunoassays and are used to detect
or measure a wide variety of serum analytes including steroid
and protein hormones, drug residues, immunoglobulins,
and pathogen antigens. They can be performed in a
number of formats with antigen or antibody absorbed onto
the plastic surface of microtiter plate wells and with primary
or secondary antibody being conjugated to a variety
of labels to allow sensitive detection. Labels that have
been used include enzymes such as horseradish peroxidase
or alkaline phosphatase, whereas more recent developments
have replaced enzyme labeling with fluorescent