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

V. Methods for Evaluation of the Immune Response to Infectious Agents
163
Positive
control
Negative
control
FIGURE 6-3 Tube agglutination test for antibodies to Brucella
canis . Serial dilutions of patient serum are compared with positive and
negative sera.
A
B
C
D
E
F
FIGURE 6-4 Microagglutination test for antibodies Toxoplasma gondii ,
performed using latex agglutination. Serum is diluted serially beginning
at 1:16.
measure a titer of antibody and require serial dilution of
the test serum. A simple positive or negative is sufficient
for some purposes, such as the test for canine rheumatoid
factor shown in Figure 6-5 .
B . Hemagglutination and
Hemagglutination-Inhibition
Some viruses have receptors for erythrocytes and when
incubated in their presence cause them to agglutinate. This
phenomenon is called hemagglutination. Specifics of the
erythrocyte source, mammalian or avian, and optimum temperature
and time for reaction vary depending on the virus
of interest. Myxoviruses, paramyxoviruses, enteroviruses,
and adenoviruses are several virus groups with members of
veterinary interest that are capable of hemagglutination. The
hemagglutination procedure is itself of little immunological
interest. However, the ability of antiserum to inhibit the hemagglutination
caused by virus receptors for erythrocytes has
been utilized to develop a serological test called hemagglutination
inhibition (HI). The antibodies bind to receptor sites
for erythrocytes and thus block the hemagglutination reaction.
The test is used to measure antibody titers to the virus.
Alternatively, with a known source of antiserum, one can use
the HI test as a preliminary step in viral identification.
To perform the hemagglutination-inhibition test, serial
two-fold dilutions of heat-inactivated serum are prepared in
saline. A 0.25-ml aliquot of each dilution is then mixed with
a similar amount of viral suspension that contains 4 hemagglutinating
units. These are mixed and incubated. Next
0.25 ml of a 1% erythrocyte suspension is added, and the
tubes are mixed again and incubated at the appropriate temperature
and time for the virus of interest. The agglutination
or absence thereof is read, and the HI titer of the serum is
assigned as the reciprocal of the highest serum dilution that
completely prevents hemagglutination. Alternatively, one
can perform the test by making serial dilutions of the virus
suspension and using a standard amount of serum. Test sera
are then compared with known negative and positive sera.
The former HI test is called the alpha procedure, and the latter
is called the beta procedure. Appropriate controls must
be included in either procedure, particularly to prevent false
positive results from the presence of hemagglutinating substances
in test sera.
C . Virus Serum Neutralization Assay
Evaluation of the protective antibody response to viral
agents is most often done using a serum virus neutralization
assay. This assay is performed by incubation of serum
dilutions with virus followed by inoculation of cell culture
with the virus/serum mixture. The cells are observed for the
development of cytopathic effect (CPE). Control cells inoculated
with virus that has been incubated with a negative
serum will show positive CPE and serve as a basis for comparison
with the test sera. Performance of dilutions allows
for the determination of a titer. Because the antibodies that
are active in this assay prevent viral entry to the cell, they
are protective. This is not necessarily true for antibodies
detected by enzyme-linked immunosorbent assay (ELISA)
or indirect immunofluorescence assay (IFA).
D. Agar Gel Double Immunodiffusion
Agar gel immunodiffusion has been used routinely to identify
horses infected with the equine infectious anemia virus
has been demonstrated by the “ gold standard ” Coggin’s
test. In this assay, serum samples from positively infected
horses are alternated with samples to be tested around the
outside wells. In the center well, EIA antigen is placed. The
development of a line showing continuity with the adjacent
lines of precipitation between viral antigen and positive
control sera (called identity) confirms that the serum
is from an infected horse. Although this assay is extremely
reliable, it is much less sensitive than other methods (such
as ELISA). Sometimes a horse newly infected will not be
positive on an initial sample but on retesting will demonstrate
the appropriate line of identity. The same horse would