Direct fluorescent antibody staining

Direct fluorescent antibody smears have become a more efficient method than Giemsa stains or tissue cultures fiar identifying chlamydia. Commercially prepared kits make specimen collection convenient, and results are available in approximately 24 hours. Good results, however, depend on obtaining an adequate specimen. Fluorescein-labeled monoclonal antibodies in the staining reagent specific for Chlamydia trachomatis outer membrane proteins bind to the C. trachomatis in the smear. Studies that compare direct fluorescein antibody techniques with tissue culture results have found acceptable sensitivity and specificity values. 

Fig. 23-13. These Yersinia pestis fluorescent cells are from infected mouse spleen. Notice how the outlines of the coccobacilli light up in this direct fluorescent antibody (DFA) test. The DFA test is specific and therefore better than the other stains discussed in this chapter (original magnification x 1,000). Photograph Courtesy of M. C. Chu, Centers for Disease Control and Prevention, Fort Collins, Colo.

Obtain a Gram stain of blood, CSF, lymph node, or sputum. Other diagnostic tests Include direct fluorescent antibody testing and PCR for antigen detection. 

Obtain blood and sputum cultures. F tularensis may be identified by direct examination of secretions, exudates, or biopsy specimens using direct fluorescent antibody or immunohistochemical stains. Serology may retrospectively confirm the diagnosis. 

The role of (1 3)-)3-D-glucanase in cell wall biosynthesis and its distribution in the mycelium of Sclerotium rolfsii have been studied. Specific zones of immunofluorescence appeared in the hyphal tips, clamp connections, new septa, and lateral branching when a specific antiserum was used with the direct method of fluorescent antibody staining. Enzyme activity in the cell wall preparation was inactivated by diethylpyrocarbonate but a majority of the activity was in a latent form which was unaffected by the ester. 

Whereas multicolor immunoenzyme staining is applicable only for separately located antigens (see Chap. 7), multicolor fluorescence immmunostaining makes it possible to colocalize antigens not only in the same cell but also in the same cellular compartment. Simultaneous immunolocalization of antigens using fluorescent antibodies can be fulfilled both by the direct (see Sect. 4.1) and indirect (see Sect. 4.2) methods. With the direct method, primary antibodies are labeled with fluorescent dyes, while with the indirect method, primary antibodies are applied as unlabeled antibodies and the visualization is performed with secondary antibodies that are labeled with fluorescent dyes. 

Figure 32.13 Expression of gustducin in the tongue. (A) A section of tongue stained with a fluorescent antibody reveals the position of the taste buds. (B) The same region stained with an antibody directed against gustducin reveals that this G protein is expressed in taste buds. [Courtesy of Dr. Charles S. Zuker.]...

Two different protocols for the direct labeling of a cell surface antigen are described next one for unfixed cells in suspension and another for fixed cells adhered to coverslips (Fig. 2). A permeabilization step (see Chapter 9) must be added for staining intracellular antigens with fluorescent antibodies or their fragments. 

As cells have not been permeabilized by this protocol, only antigen domains accessible from the outer side of the plasma membrane will be stained. When a directly fluorescently conjugated first antibody is used. Protocol 2D, steps 4 and 5 are omitted. 

Fluorochromes are stains that interact directly with cellular components, or are used to form conjugates with antibodies or ligands, yielding fluorescent reporter molecules. O Table 13-1 lists a selection of fluoro- D Table 13-1 Fluorochromes for flow cytometry ... 

The actual response of monoclonal antibodies with individual cells is usually visualized either directly (typically using fluorescent stains) or indirectly [using the reaction of antibody labeled with horseradish peroxidase (HRP) or other enzymes] with diaminobenzidine (DAB) (or other substrate while using other enzymes) under the microscope or in the flow cytometer. The latter, however, is not employed routinely in CSF immunocytology, although it has an advantage in clinical hematology. 

BrdU/DNA flow cytometry offers flexibility and diversity in the study of cell kinetics from cells in culture to human tumors in vivo. The essence of the procedure is to pulse label with BrdU by a short-term incubation in vitro or by a single injection in vivo samples are then taken at time intervals thereafter and stained after fixation in ethanol. The cells are then stained with a monoclonal antibody against BrdU that can be either directly conjugated to a fluoro-chrome (usually fluorescein isothiocyanate [FITC]) or, alternatively, bound to a second antibody conjugated with FITC. The cells are then counterstained with propidium iodide (PI) to measure the DNA content and analyzed on the flow cytometer. The results are displayed as linear-red fluorescence on the x-axis vs linear or log-green fluorescence on they-axis. 

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