In the diagnosis of infectious diseases, immunologic methods take advantage of the specificity of antigen–antibody binding. For example, known antigens and antibodies are used as diagnostic tools in identifying microorganisms. In addition, serologic detection of a patient’s immune response to infection, or antigenic or nucleic acid evidence of a pathogen in a patient’s body fluids, is frequently useful. Immunologic methods are useful when the infecting microorganism is difficult or impossible to isolate or when a previous infection needs to be documented. Most methods for determining whether antibodies or antigens are present in patients’ sera or other body fluids require some type of immunoassay procedure such as those described in this section.

A. Detection of microbial antigen with known antiserum

These methods of identification are often rapid and show favorable sensitivity and specificity. However, unlike microbial culturing techniques, these immunologic methods do not permit further characterization of the microorganism, such as determining its antibiotic sensitivity or characteristic metabolic patterns.

1. Quellung reaction: Some bacteria having capsules can be identified directly in clinical specimens by a reaction that occurs when the organisms are treated with serum containing specific anti bodies. The Quellung reaction makes the capsule more refractile and thus more visible, but the capsule does not actually swell. This method can be used for all serotypes of S. pneumoniae, H. influenzae type b, and Neisseria meningitidis groups A and C.

 2. Slide agglutination test: Some microorganisms, such as Salmonella and Shigella species, can be identified by agglutination (clumping) of a suspension of bacterial cells on a microscopic slide. Agglutination occurs when a specific antibody directed against the microbial antigen is added to the suspension, causing cross-linking of the bacteria.

 B. Identification of serum antibodies

 Detection in a patient’s serum of antibodies that are directed against microbial antigens provides evidence for a current or past infection with a specific pathogen. A discussion of the general interpretation of antibody responses includes the following rules: 1) antibody may not be detectable early in an infection, 2) the presence of antibodies in a patient’s serum cannot differentiate between a present and a prior infection, and 3) a significant rise in antibody titer over a 10 to 14-day period does distinguish between a present or prior infection. Techniques such as complement fixation and agglutination can be used to quantitate antimicrobial antibodies.

1. Complement fixation: One older but still useful method for detecting serum antibody directed against a specific pathogen employs the ability of antibody to bind complement (Figure 1). A patient’s serum is first incubated with antigen specific for the suspected infectious agent, followed by the addition of complement. If the patient’s serum does contain immunoglobulin (Ig) G or IgM antibodies that target the specific antigen (indicating past or current infection), then the added complement will be sequestered in an antigen–antibody–complement complex (“complement fixation”). Next, sensitized (antibody-coated) indicator sheep RBCs are added to the solution. If complement has been fixed (because the patient’s serum contained antibodies against the added anti gen), then little complement will be available to bind to the antibody–RBC complexes, and the cells will not lyse. If complement has not been depleted by initial antigen–antibody complexes (because the patient’s serum does not contain antibodies to the specific antigen), the complement will bind to the antibody–RBC complexes, causing the cells to lyse. As hemolyzed RBCs release hemoglobin, the reaction can be monitored with a spectrophotometer.

Fig1. Complement fixation.

 2. Direct agglutination: Direct bacterial agglutination testing is sometimes ordered when a suspected pathogen is difficult or dangerous to culture in the laboratory. This test measures the ability of a patient's serum antibody to directly agglutinate specific killed (yet intact) microorganisms. This test is used to evaluate patients suspected of being infected by Brucella abortusor Francisella tularensis, among others.

 3. Direct hemagglutination: Antibodies directed against RBCs can arise during the course of various infections. For example, such antibodies are typically found during infectious mononucleosis caused by Epstein-Barr virus . When uncoated (native) animal or human RBCs are used in agglutination reactions with serum from a patient infected with such an organism, antibodies to RBC antigens can be detected. The patient’s anti bodies cause the RBCs to clump. This test is, therefore, a direct hemagglutination reaction. In the case of some diseases, including pneumonia caused by Mycoplasma pneumoniae, IgM autoantibodies may develop that agglutinate human RBCs at 4oC but not at 37oC. This is termed the “cold agglutinins "test.

 C. Other tests used to identify serum antigens or antibodies

 1. Latex agglutination test: Latex and other particles can be readily coated with either antibody (for antigen detection) or antigen (for antibody detection). Addition of antigen to antibody-coated latex beads causes agglutination that can be visually observed (Figure 2). For example, such methods are used to rapidly test CSF for antigens associated with common forms of bacterial or fungal meningitis. When antigen is coated onto the latex bead, antibody from a patient’s serum can be detected. Latex agglutination tests are widely used for the identification of β-hemolytic streptococci group A.

Fig2. A. Schematic representation of antigens agglutinating latex beads with bound antibody. B. Photograph of agglutination reaction.

 2. Enzyme-linked immunosorbent assay: Enzyme-linked immunosorbent assay (ELISA) is a diagnostic technique in which antibody specific for an antigen of interest is bound to the walls of a plastic microtiter well (Figure 3). Patient serum is then incubated in the wells, and any antigen in the serum is bound by the antibody on the well walls. The wells are then washed, and a second antibody is added. This one is also specific for the antigen but recognizes epitopes different from those bound by the first anti body. After incubation, the wells are again washed, removing any unattached antibody. Attached to the second antibody is an enzyme, which, when presented with its substrate, produces a colored product, the intensity of the color produced being proportional to the amount of bound antigen. ELISAs can also be used to detect or quantitate antibody in a patient’s serum. In this instance, the wells are coated with antigen specific for the antibody in question. The patient’s serum is allowed to react with the bound antigen, the wells are washed, and a secondary antibody (that recognizes the initial antibody) conjugated to a color product–producing enzyme is added to the well. After a final washing, substrate for the bound enzyme is added to the well, and the intensity of the colored product can be measured.

Fig3. Principle of enzyme-linked immunosorbent assay (ELISA).

3. Fluorescent-antibody tests: Organisms in clinical samples can be detected directly by specific antibodies coupled to a fluorescent compound such as fluorescein. In the direct immunofluorescence antibody technique, a sample of concentrated body fluid (for example, CSF or serum), tissue scraping (for example, skin), or cells in tissue culture is incubated with a fluorescein-labeled antibody directed against a specific pathogen. The labeled antibody bound to the microorganism absorbs ultraviolet light and emits visible fluorescence that can be detected using a fluorescence microscope. A variation of the technique, the indirect immunofluorescence anti body technique, involves the use of two antibodies. The first is unlabeled antibody (the target antibody), which binds a specific microbial antigen in a sample such as those described above. This clinical sample is subsequently stained with a fluorescent antibody that recognizes the target antibody. Because a number of labeled antibodies can bind to each target antibody, the fluorescence from the stained microorganism is intensified.

مواضيع ذات صلة

Identification of Bacteria

Growing Bacteria in Culture

Cultivation of Listeria, Corynebacterium, and Similar Organisms

Pathogenesis and Spectrum of Disease for Listeria, Corynebacterium, and Similar Organisms

The Growth Curve in Batch Culture

The Measurement of Microbial Concentrations

Bacterial Pathogenesis

Distribution of Normal Flora in The Body

Cultivation of Bacillus and Similar Organisms

Diagnostic Laboratory Tests for Borrelia Burgdorferi

Bacterial Cell wall

Bacillus cereus