Immunofluorescent assays are frequently used for detecting bacterial and viral antigens in clinical laboratories. In these tests, antigens in the 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 applied to the specimen. After appropriate incubation, washing, and counterstaining (staining of the back ground 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 (FITC) as the fluorescent dye. FITC fluoresces a bright apple-green (Figure 1).
Fig1. Legionella (Direct) Fluorescent Test System (Scimedx Corp., Denville, N.J.). Legionella pneumophila serogroup 1 in sputum.
Fluorescent antibody tests are performed using either a direct fluorescent antibody (DFA) or and indirect fluorescent antibody (IFA) technique (Figure 2). In the DFA technique, FITC is conjugated directly to the specific antibody. In the IFA technique, 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 to the FITC. The IFA is a two-step, or sandwich, technique. The IFA technique is more sensitive than the DFA method, although the DFA method is faster because it involves a single incubation.
Fig2. Direct and indirect fluorescent antibody tests for antigen detection.
The major advantage of immunofluorescent micros copy assays is the ability to visually assess the adequacy of a specimen. This is a major factor in tests for the identification of chlamydial elementary bodies or respiratory syncytial virus (RSV) antigens. Microbiologists can discern whether the specimen was collected from the columnar epithelial cells at the opening of the cervix in the case of the Chlamydia DFA test or from the basal cells of the nasal epithelium in the case of RSV. Reading immunofluorescent assays requires extensive training and practice for laboratory personnel to become proficient. Finally, fluorescence dyes fade rapidly over time, requiring digital imaging to maintain archives of the results. For this reason, some antibodies have been con jugated to other markers instead of fluorescent dyes. These colorimetric labels use enzymes, such as horseradish peroxidase, alkaline phosphatase, and avidin-biotin, to detect the presence of antigen by converting a color less substrate to a colored end product. The advantage of these tags is that they allow the preparation of permanent mounts, because the reactions do not fade with storage and visualization does not require a fluorescent microscope.
In clinical specimens, fluorescent antibody tests are commonly used to detect infected cells that harbor Bordetella pertussis; T. pallidum; L. pneumophila; Giardia, Cryptosporidium, Pneumocystis, and Trichomonas spp.; herpes simplex virus (HSV), cytomegalovirus, varicella-zoster virus, RSV, adenovirus, influenza virus, and parainfluenza virus.
