Immunofluorescent Assays

Immunofluorescent assays are popular tests for detecting antigens in clinical laboratories. In these tests, antigenic determinants in patient specimens are immobilized and fixed onto glass slides with formalin, methanol, ethanol, or acetone. Monoclonal or polyclonal antibodies conjugated (attached) to fluorescent dyes are then applied to the specimen. After appropriate incubation, washing, and counterstaining (staining the background with a nonspecific fluorescent stain such as rhodamine or Evan's blue), the slide is viewed using a microscope equipped with a high-intensity light source (usually halogen) and filters to excite the fluorescent tag. Most kits used in clinical microbiology laboratories use fluorescein isothiocyanate (FTTC) as die dye; FTTC fluoresces a bright apple-green (Figure 9-10).

Fluorescent antibody tests are performed using either a direct (DFA) or indirect (IFA) technique (Figure 9-11). In the DFA, FITC is conjugated directly to the specific antibody. In the IFA, the antigen-specific antibody is unlabeled and a second antibody (usually raised against the animal species from which the antigen-specific antibody was harvested) is conjugated

Direct Immunofluorescence Digramof
Figure 9-9 Diagram of liposome-latex agglutination reaction.
Figun 1-10 Legionella (Direct) Fluorescent Test System, Scimedx Corp., Denville, NJ. Legionella pneumophila serogroup 1 in sputum.

Fluorescent

Fluorescent

Secondary

Indirect

Figure 9-11 Direct and indirect fluorescent antibody tests for antigen detection.

Indirect

Figure 9-11 Direct and indirect fluorescent antibody tests for antigen detection.

to the FTTC. The IFA is a two-step, or sandwich, procedure. IFA is more sensitive than DFA, although DFA is faster because there is only one incubation step.

The major advantage of immunofluorescent microscopy assays is that they allow visual assessment of the adequacy of a specimen. This is a major factor in their use in tests for chlamydial elementary bodies or respiratory syncytial virus (RSV) antigens. Microscopists can see if the specimen was taken from the columnar epithelial cells at the opening of the cervix in the case of the chlamydia DFA, or from the basal cells of the nasal epithelium in the case of RSV. Many individuals, however, consider it problematic that reading slides is completely subjective and that microbiologists must have extensive training to perform testing. Likewise, many individuals view the requirement for a fluorescent scope as an expensive luxury. Finally, fluorescence fades rapidly over time, which makes the archiving of slides difficult. Therefore, antibodies have been conjugated to other markers besides fluorescent dyes. These newer colorimetric labels use enzymes, such as horseradish peroxidase, alkaline phosphatase, and avidin-biotin, to detect the presence of antigen by converting a colorless substrate to a colored end product. Advantages of these tags are that they allow the preparation of permanent mounts because the reactions do not fade with storage and they can be detected using a simple light microscope.

Fluorescent antibody tests are commonly used to detect Bordetella pertussis, Legionella pneumophila, Giardia, Cryptosporidium, Pneumocystis, Trichomonas, herpes simplex virus, cytomegalovirus, varicella-zoster virus, RSV, adenovirus, influenza virus, and parainfluenza virus in clinical specimens.

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Bacterial Vaginosis Facts

Bacterial Vaginosis Facts

This fact sheet is designed to provide you with information on Bacterial Vaginosis. Bacterial vaginosis is an abnormal vaginal condition that is characterized by vaginal discharge and results from an overgrowth of atypical bacteria in the vagina.

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