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

V. Methods for Evaluation of the Immune Response to Infectious Agents
167
FIGURE 6-10 (a) Schematic demonstrating
direct and indirect ELISA. Direct ELISA measures
antigen-specific antibody; indirect ELISA measures
antigen. (b) Photograph of 96 well ELISA
plate: darkest color indicates most positive reaction.
Quantitation is performed by measuring optical
density of well contents.
(a)
Direct
Indirect
(b)
chain-specific antisera as detection reagents. The appearance
of both direct and indirect ELISA in which intense color
equates with a positive result is shown in Figure 6-10b . A
less commonly used configuration of ELISA is the competitive
ELISA. This type of ELISA can be constructed to detect
either antigen or antibody. It utilizes an enzyme-labeled
ligand (antigen or antibody), which then competes with its
unlabeled counterpart in the patient’s serum. Such an assay
shows color (higher O.D.) when the sample is negative.
Thus, it is important for those interpreting ELISA to fully
understand what constitutes a negative and a positive sample.
The use of appropriate controls makes this of minor concern.
H . Immunohistochemistry/
Immunoperoxidase Techniques
Just as immunofluorescence can be used to evaluate serum
antibodies or antigen present in tissue or on cells, the technique
of enzyme immunoassay is also applied to tissue
sections for identification of cell populations and demonstration
of antigens in tissues. Immunoperoxidase detection
of antigens has the advantage that the tissue sections
can be examined with a standard microscope, whereas
the immunofluorescence-based testing requires utilization
of mercury lamps, dichroic mirrors, and special filters for
excitation of the fluorochrome and for visualization of the
emitted fluorescence. For immunoperoxidase evaluation of
tissue antigens, an enzyme tagged antiserum is used to bind
the antigen, then the substrate for the enzyme is added. The
substrate is chosen so that it changes color when hydrolyzed
by the enzyme. Thus, a colored product is deposited
permanently in the tissue. In general, direct immunofluorescence
and immunohistochemistry can be used for the
same type of determinations. Figure 6-11 shows a canine
lung section infected with canine distemper virus using
immunoperoxidase staining with anti-canine distemper
virus (CDV) antibody.
I . Western Blot Analysis
Western blot immunoassay is performed when it is desirable
to determine which antigens in a mixture are binding
with antibodies in test sera. It has great value when it is
necessary to discriminate between antibodies produced in
response to vaccination and those produced as a result of
infection. If a subunit vaccine is used, this technique can
be successfully applied. Some of the newer “ designer ” vaccines
use only one or two protective epitopes to immunize.