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

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Chapter | 6 Clinical Veterinary Immunology
FIGURE 6-1 Single radial immunodiffusion test
for determination of IgA levels in serum.
A
Standards 1-5
Various patient’s serum
B
C
1
2 3 4 5 6
(a)
(b)
(c)
FIGURE 6-2 (a) Normal serum immunoelectrophoresis (IEP) showing
IgG, IgM, and IgA. (b) IEP showing lack of gamma globulins. (c) IEP
demonstrating an IgA myeloma protein in patient’s serum.
This technique is performed by incubating peripheral blood
lymphocytes with mitogens or specific antigen for several
days. The addition of tritiated thymidine or a nonradioactive
dye that incorporates into dividing cells provides a signal
that is commensurate with the degree of cell division.
Comparison of the stimulated cells with unstimulated cells
from the same animal provides a stimulation index, which
indicates the amount of responsiveness inherent in the
cells of the patient. Usually the assay is used to determine
whether or not a patient has suppressed or diminished T
cell function in general. In can be used to measure the T
cell response to specific antigen.
Multicolor flow cytometry can be used to demonstrate
the presence of T cells making either Th1 or Th2 cytokines.
This technique utilizes surface staining to distinguish CD4
or CD8 T cell populations followed by permeabilization,
incubation with a reagent that prevents newly synthesized
protein from leaving the Golgi apparatus, and staining for
intracellular cytokine using antibodies conjugated with different
fluorochromes.
V . METHODS FOR EVALUATION OF THE
IMMUNE RESPONSE TO INFECTIOUS
AGENTS
A . Agglutination and Passive Agglutination
The antibody response to a specific antigen can be evaluated
by a variety of assays. For a particulate antigen, an agglutination
test can be performed. In this test, serum is serially
diluted, and the dilutions are then mixed with an antigen
suspension. The tubes are observed for agglutination. The
titer is the inverse of the dilution in the last tube that shows
a positive agglutination response. This is a traditional assay
that is still used for detection of antibodies to a variety
of bacterial antigens. It is possible to use this method to
evaluate titers for soluble antigens by binding them to latex
particles. Figure 6-3 shows a tube agglutination test used
to measure the titer of antibodies against Brucella canis in
canine serum. When a soluble antigen is linked to a particle,
it becomes a passive agglutination test. The microagglutination
test for antibodies to Toxoplasma gondii is
performed this way. Figure 6-4 shows an example of the
Toxotest, run in microtiter plate wells. Both of these assays