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

V. Methods for Evaluation of the Immune Response to Infectious Agents
165
FIGURE 6-6 Direct immunofluorescence demonstrates
herpesvirus in a feline conjunctival smear.
Arrow shows fluorescent intracellular virus.
FIGURE 6-7 Direct immunofluorescence demonstrates
deposition of IgG in a glomerulus of
a dog with immune complex mediated kidney
disease.
a pathogen is intracellular and can easily be propagated
in culture. Indirect immunofluorescence is performed to
detect and quantitate titer of antibodies specific for these
cell-associated antigens. When IFA is used, it is not necessary
to purify the antigen. Cell-associated antigen, such as
pathogens that are intracellular or nuclear antigen (in the
case of the antinuclear antibody test), are present in cells
that are fixed onto slides. The serum to be evaluated is then
diluted serially and applied to wells containing the cells.
The application of a fluorescein-conjugated secondary
antibody then allows binding of the conjugate to antibodies
present in the serum that have recognized epitopes present
on the antigen. Several washing steps must be properly
performed for this technique to yield reliable information.
The technique of direct immunofluorescence also has
diagnostic application. In direct immunofluorescence, the
sample comes from the patient and consists of cells or tissues
rather then serum. A known fluorochrome-conjugated
serum is used to detect the antigen of interest. For example,
conjunctival cells or transitional epithelial cells from the
bladder of canine distemper-infected dogs can be stained
with FITC-antidistemper antibodies to demonstrate viral
infection. Another application of this technique involves
diagnosis of autoimmune disease and is included under the
discussion of autoimmunity. Figure 6-6 shows conjunctival
cells from a cat infected with herpesvirus (direct FA).
For detection of IgG or C3, deposition in blood vessels
of patient’s that have immune complex disease direct FA
is often used. Deposition of IgG in a kidney glomerulus is
shown in Figure 6-7 . Indirect immunofluorescence is used
to demonstrate the presence and measure the titer of antinuclear
antibodies, as shown in Figure 6-8 .