Direct fluorescent antibody method

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. 

Diagnosis and Treatment Skin anthrax may be diagnosed from the biopsy of the sore and performing microscopic examination of the organism. Inhalation anthrax however, is difficult to diagnose. Chest x-ray, lab cultures and blood tests should be carried out. Rapid laboratory tests may be carried out to diagnose anthrax. Such tests include polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and direct fluorescent antibody (DFA) methods. 

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. 

Heterogenous fluorescence immunoassays can be carried out with the aid of the same separation procedures used in radioimmunoassay. The more expedient homogenous fluorescence immunoassays require quenching, enhancement, polarization, or shifting of the fluorescence of the label upon binding of the labeled analyte to its antibody. Occasionally, a second antibody, directed at the anti analyte antibody, will be used in a double-antibody method to precipitate the bound labeled and unlabeled analyte or to alter the optical properties of the label in such a way as to make the analysis more sensitive. 

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. 

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