Antigen Detection Assays

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 antigenic determinant.

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, latex agglutination, 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.

Immunofluorescence
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 fluorescent antibody (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 the microscope. 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.
Latex Agglutination
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.