IMMUNOFLUORESCENT ASSAYS

written by Wendy Niefer Much of the information in the immunoassay section was obtained from the following references, and the student is encouraged to use reference sources for more details than are given here.

1. Lennette, E. H. et al. Manual of Clinical Microbiology, 4th ed. American Society for Microiology, Washington, D. C. 1985.

2. Rose, N. R. and Friedman, H. et al. Manual of Clinical Immunology, 2nd ed. American Society for Microbiology, Washington, D. C. 1980.

3. Rose, N. R. et al. Principles of Immunology, 2nd ed.MacMillan Publishing Co. Inc., New York 1979

Immunoglobulins and many antigens can be coupled to fluorescent dyes. The reactions of these antigens or antibodies can be made visible in histological or cytological preparation with a fluorescence microscope. Fluorescent dyes or fluorochromes are a group of substances that achieve elevated but unstable energy levels by absorbing light at a certain wavelength and immediately emitting light at another wavelength. The most widely used fluorochromes in IF studies are fluorescein and rhodamine. They are conjugated to a specific antibody (direct method) or to an anti-species antibody (indirect method). Sensitivity of IF is dependent on the immune properties of the reagents and also on the optical system employed. IF methods of studying ag/ab reactions have three advantages. First, these methods make it possible to locate antigens in histological or cytological preparations.

Second, because whole cells or tissue sections may be examined, most or all of their antigenic components are available as reaction sites.

Third, a variety of test procedures exists for the detection of antigen, antibodies, or complement. In bacteriology, IF is usually used to detect bacterial cells in specimens or to confirm the identification of an isolate. These assays are called fluorescent antibody (FA) stains and are usually done by the direct method. Direct FA conjugates are available for B. pertussis, some Enterobacteracae, Legionella sp., N. gonorrhoea and N. meningitidis, Chlamydia sp., and Treponema pallidum. Antibodies to treponema (syphilis) are detected by an indirect FA test.

Cautions:

The purity of the antibody/conjugate is the most important consideration in IF procedures. Reagents are available commercially for most tests, but not for some specialized applications. IF assays are less sensitive than radioimmunoassays or enzymeimmunoassays, but is more sensitive than precipitation. Over-washing of stained preparations can move some soluble antigens to other tissue sites. Background staining or autofluorescence can interfere.

The methods that are covered in this section include

Counterimmunolelectrophoresis

Precipitation

Agglutination

ImmunoFluorescent Assays

Enzyme Immuno Assays

Neutralization

NonSpecific Immuno Assays

Bacterial infections usually result in an immune response in the host. The products of the immune response react specifically with the bacterial immunogens that stimulated their production. Assays have been developed that use bacterial antigens to detect the antibodies produced by the immune system and antibodies to detect antigens.

The immune response of the host to some bacterial antigens may result in specific immunity to, or recovery from, a particular infection. Antibodies to other bacterial antigens do not effect the course of the disease nor do they provide immunity. They are important for use in immunoassay procedures. This section will deal with the uses immunology has been put to in the bacteriology laboratory under three major headings:

1.Detection of Bacterial Antigens; 2.Response to Bacterial Antigens and; 3.Principles of Specific Immunoassays. There are advantages and disadvantages to the use of antigens and antibodies as reagents.

Advantages: The specificity of antibodies for antigens in reactions means little interference from other substances. A wide range of choices exists for procedures depending on the sensitivity required. Manual manipulation is usually simple, but most methods have the ability to be semi- and fully automated with easily obtained and inexpensive equipment. Reagents are inexpensive with a long shelf life and are chemically non-toxic. A variety of labels may allow multiple, simultaneous assays. Some assays are quicker than culture and do not rely on viable organisms.

Disadvantages; A biological hazard may exist depending on source of reagent (usually sera; eg. Hepatitis B). The stability of the antigen/antibody reaction may require critical control of some variable conditions like temperature or timing. The purity of antibody or antigen, cross-reactivity (sometimes advantageous), or presence of haptens (incomplete antigens) must be established. Expertise is required in the manufacture and labeling procedures to ensure the quality of the reagents. A number of control procedures are usually required to check reactivity and monitor interference from non-specific or non-immunologic reactions. Some procedures are slow, but frequently another assay technique can be employed. The procedures should not be relied upon to replace culture techniques.