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

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Chapter | 6 Clinical Veterinary Immunology
FIGURE 6-11 Immunoperoxidase staining of
dog lung infected with canine distemper virus.
Brown staining indicates the presence of intracellular
virus as shown with horseradish peroxidase
conjugated anti-CDV and substrate.
In this case, it is possible to evaluate serum using the
Western blot method to identify to which of multiple proteins
present in a pathogen a patient has responded. For
example, in the vaccine for Lyme disease, if a dog has
bands on Western blot for numerous Borrelia burgdorferi
proteins, it is likely to be infected. However, the presence
of only antibodies to Osp A (the protein containing protective
epitopes that is present in the vaccine) indicates a
vaccinated uninfected dog. The Western blot has also been
used to differentiate between Sarcocystis neurona exposed
and infected horses in patients suspected of having equine
protozoal myelitis (EPM). Figure 6-12 shows an EPM
Western blot.
To perform a Western blot assay, the antigen preparation
must first be separated into its protein components
using polyacrylamide gel electrophoresis (PAGE). This
technique denatures the disulfide bonds and causes proteins
to move through the gel according to their molecular
weight. After the antigen is separated, the gel is blotted
electrophoretically onto a nitrocellulose membrane,
which is then probed with the patient sera and ultimately
an enzyme-conjugated secondary antibody and substrate
to visualize the bands representing proteins recognized by
antibodies in the patient’s serum. It is possible, by using
specific conjugated antisera, to identify which antibody
isotypes are binding (IgM, IgG, IgA, or IgE).
J . How Do the Sensitivities of Different
Immunoassays Compare
Generally the gel diffusion-based assays such as double
and single radial diffusion are much less sensitive that the
primary binding assays like ELISA. This is because the
former require not only binding of antigen to antibody but
also the formation of an appropriate size insoluble precipitate
for detection visually. Recently an ELISA kit has
been developed to test for antibodies to equine infectious
anemia. The traditional gold standard EIA assay is based
Negative
control
Positive
control
CSF CSF serum
FIGURE 6-12 Western blot for equine protozoal myelitis. Sarcocystis
neurona antigen was separated by SDS-PAGE, blotted to nitrocellulose,
and probed with either sera or CSF. Bands on positive and negative control
strips are compared to those on test strips.
on gel diffusion. The use of these two tests represents an
interesting comparison in sensitivity between test methods,
as the gel diffusion-type tests are far less sensitive than the
primary binding assays like ELISA. Generally a negative
ELISA for EIA is an acceptable result, but a positive is
tested again by the Coggin’s test to avoid false positives in
the diagnosis of this important reportable equine infectious
disease. The agglutination assays are generally between the
gel diffusion and ELISA-type assays in sensitivity, and the
immunofluorescence assays are less sensitive than ELISA
but more sensitive than the gel diffusion-type assays.
These comments are generalities, and each test will depend
on the quality of the reagents used for both sensitivity and
specificity.