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

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Chapter | 6 Clinical Veterinary Immunology
FIGURE 6-8 Indirect immunofluorescence
demonstrates the presence of antinuclear antibodies
in serum of a dog with systemic lupus erythematosus.
Arrow points to nuclear fluorescence in
HEP-2 cells; arrowhead shows nonfluorescence of
cytoplasm.
F . Flow Cytometry
Flow cytometry uses fluorochromes conjugated to specific
antisera to identify and quantitate cells of various types.
This is an application of immunofluorescence. It is most
commonly used to identify populations of cells, such as T
cells and dendritic dells. The availability of antisera specific
for activation markers, such as CD25 (IL-2 receptor),
makes it possible to evaluate activated versus resting cells.
This technique has broad application for diagnosis of a variety
of immunological, neoplastic, and infectious diseases.
Diagnosis of viral immunodeficiency (SAIDS, FIV) often
utilizes flow cytometry to determine ratios of CD4 to
CD8 cells. An example of data from flow cytometry is
shown in Figure 6-9 .
G . Enzyme-Linked Immunosorbent Assay
(ELISA)
Enzyme-linked immunosorbent assay (ELISA) is the primary
binding assay that has become a standard for many diagnostic
tests since the 1980s. It is used both as a quantitative assay
for serum antibodies to a variety of antigens and as a quick
test for a simple positive or negative answer to the question
of whether or not a patient is infected with a particular pathogen.
Because the ELISA is a primary binding assay, it is
very sensitive. The specificity of each ELISA depends on the
quality and specificity of the reagents used. The technique
has the ability to be both very specific and very powerful if
appropriately configured. There are two main configurations,
one for detection of antibody and the other for detection of
antigen ( Fig. 6-10a ). In the indirect form of ELISA, antigen
is detected using a “ catching ” antibody attached to a solid
substrate, most often a microtiter plate. The sample is added
38.4
4.57
33.6 23.4
FIGURE 6-9 Plot from flow cytometry analysis. The plot shows cells
stained for CD4 and CD8. The y-axis shows cells stained for CD4 with
the fluorochrome FITC, and the x-axis shows cells stained to detect
CD8 cells with another fluorochrome, Alexa 647. These cells are from
bovine afferent lymph. This sample has 38.4% CD4 cells and 23.4%
CD8 cells. A population of cells (33.6%) is neither CD4 nor CD8 cells.
and it is detected after a series of incubation and wash steps
by another antibody specific for the antigen, either conjugated
with an enzyme, or another variation, such as biotin
(which is followed with addition of an enzyme attached to
avidin). Addition of the substrate for the enzyme produces
a colored product, whose optical density is determined by
spectrophotometry. In the direct format, the antigen coats
the solid substrate and serum is added to that. Antibodies
present bind the antigen and are then detected by specific
antisera recognizing the species-specific antibody. This technique
has the advantage of allowing for selection of a particular
class of antibody, such as IgM or IgE, by using heavy