All microorganisms contain a variety of different antigens whose composition is usually protein
or carbohydrate in nature. Antigens may be components of the microbial cell wall, capsule,
or intra- or extracellular enzymes. In the animal body, these antigens are recognized as
foreign substances by the host immune system, which responds by producing specific protein
molecules called antibodies
. Antibodies bind specifically with the antigen that elicited
their production. For example, the antigenic carbohydrate
capsule of Streptococcus pneumoniae
, which binds with its specific antibody in the quellung reaction.
The use of an antibody to detect the presence of a specific microbial antigen is
called an immunoassay
. The sensitivity of immunoassays depends on the quality of the antibody
preparation. In the early development of immunoassays, the antibody preparations were
not pure enough to react only with a specific antigen (known as an antigenic determinant)
on a specific microorganism. The result was often a false-positive reaction
, in which a microorganism
other than the one being tested for was detected because they shared a common
Through developments in immunology, a more specific type of antibody known
as a monoclonal
antibody can now be produced in large quantities. In contrast to the previously
used polyclonal antibodies, monoclonal antibodies react with antigenic determinants
that are unique to one microorganism and not shared by others. As a result, false-positive test
reactions are greatly reduced and a wide variety of antigen-detection tests can now be performed
in the clinical microbiology laboratory.
Many immunoassays are available, but three major types are in common use: immunofluorescence,
and enzyme immunoassay
or EIA. For these immunoassays,
monoclonal antibodies are labeled with (attached to) a “marker” molecule that provides a
means of detecting whether an antigen-antibody reaction has taken place. Polyclonal antibody
preparations are sometimes used for special purposes. The principles of these three assays
are described briefly.
In immunofluorescence assays, antibodies are labeled with a fluorescent dye called fluorescein.
When the antibodies combine with their specific antigen in a preparation, the bright
fluorescence can be visualized with a fluorescence microscope fitted with an ultraviolet illuminator
and special filters. The test is performed by placing a smear of a clinical sample on a
microscope slide and fixing it with a suitable reagent. In the simplest method, known as a direct
(DFA) test, the fluorescein-labeled antibody preparation is applied directly
to the specimen slide, which is then incubated, washed, and viewed under the fluorescence
microscope. A positive test is indicated by the presence of brightly fluorescing
organisms in the preparation (see colorplate 23, 40 and 53
For the indirect fluorescent antibody
(IFA) test, two antibody preparations are needed.
The first, which is not labeled with the fluorescent dye, contains antibodies against the microbial
agent we wish to detect. If the agent is present in the specimen smear, an antigenantibody
reaction occurs. To detect this combination, the second antibody, labeled with fluorescein,
is applied to the preparation. This second antibody has been prepared to react with
the first, unlabeled antibody. Again, a positive result is indicated by bright fluorescence under
Figure 19.1 illustrates the principle of direct and indirect fluorescence assays.
Fluorescent antibody tests are in widespread use to diagnose infections caused by a variety of
microbial agents including bacteria, viruses, and protozoa.
In latex agglutination assays, antibodies are attached to latex (polystyrene) beads that serve as
the marker for detecting the antigen-antibody interaction. Each latex particle is about 1 m
in diameter and can be charged with thousands of antibody molecules. Antibody-coated latex
particles form a milky suspension, but when they are mixed with a preparation containing
specific antigen, the resulting antigen-antibody complex results in visible clumping.
Figure 19.2 illustrates the events associated with the latex particle agglutination reaction.
Latex agglutination tests are usually performed on a glass slide or a specially treated
cardboard surface using small volumes of latex particles and liquid clinical sample. The
reagent is mixed with the clinical sample using a stirrer, and the slide is rocked by hand or
rotated with a mechanical device for several minutes before being examined visually for
clumping of the latex particles. Colorplate 24
illustrates the appearance of positive and negative
latex agglutination slide tests.
In clinical laboratories, latex agglutination tests are used to detect soluble microbial
antigens directly in serum or cerebrospinal fluid specimens, or for identifying various
types of bacteria recovered from culture plates.
|Figure 19.1 (a) In the direct fluorescent antibody (DFA) test, antibody specific for the microorganism sought is conjugated with the dye fluorescein. The antibody preparation is added to a specimen fixed to a slide. If the specific microorganism is present, the preparation will fluoresce when viewed under a fluorescence microscope. (b) In the indirect fluorescent antibody (IFA) test, the antibody specific for the microorganism is not conjugated with the dye, but will bind to the specific microorganism on the slide. A second antibody preparation, labeled with fluorescein, has been prepared to react with the first, unlabeled antibody and will fluoresce when viewed microscopically.