Fluorescent antibody staining

Antisera against poly(ADP-ribose) were raised in rabbits [8] and affinity-purified antibodies from y-globulin were obtained [9]. We routinely examined antibodies against poly(ADP-ribose), dsDNA or sin e-stranded DNA (ssDNA) by an enzyme-linked immunosorbent assay (ELISA) [10]. Nine serum samples from SLE patients with high titers against all or any of these nucleic acids were selected for fluorescent antibody-staining for poly(ADP-ribose). 

Fluorescent antibody staining Procedure in fluorescence microscopy that uses a fluoro-chrome attached to antibodies to detect the presence of an antigen. 

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. 

As already indicated adsorption, penetration, and uncoating of AAV do not require helper virus and purified DNA is infectious but requires helper. However, in the absence of helper no DNA or RNA synthesis is detected. Therefore, a helper function (s) appears to be required to initiate AAV-specific nucleic acid synthesis. Co-infection with herpes simplex virus type 1 (HSV-1) also serves to allow AAV DNA or RNA synthesis (Boucher et al., 1971 Rose and Koczot, 1972). The extent of AAV nucleic acid synthesis appeared to be comparable with either HSV or adenovirus as the helper. AAV DNA synthesized in the presence of HSV has been reported to be infectious (Boucher et al., 1971). Additionally, AAV antigens in cells co-infected with HSV are detectable by means of a fluorescent antibody staining technique (Atchison, 1970 Blacklow et al, 1970 Blacklow et al, 1971 Johnson et al., 1972). However, no infectious particles can be recovered with HSV as the helper. Interestingly, antisera to both AAV-specific SDS-polypeptides and virion protein stain the infected cells. 

Goldstein, G. Slizys, I. S. Chase, M. W. Studies on fluorescent antibody staining. I. Nonspecific fluorescence with fluorescein-coupled sheep antirabbit globulins. J. Exp. Med. 1961, 114, 89-110. 

Mikhailov, I. F. and Stanislavskii, E. S. (1963). Staining isolated bacterial structures with fluorescent antibodies. Science 40, 74-9. 

Laboratory findings include leukocytosis with predominance of mature and immature granulocytes in 50% to 75% of patients. Because L. pneumophila stains poorly with commonly used stains, routine microscopic examination of sputum is of little diagnostic value. Fluorescent antibody testing can be performed to diagnose Legionnaires disease. 

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. 

It was shown by Creech and Jones (1) in 1940 that proteins, including antibodies, could be labeled with a fluorescent dye (phenylisocyanate) without biological or immunological effects to the intended target. In theory, fluorescent reporters (tracers, probes, antibodies, stains, and so on) can be used to detect or measure any cell constituent, provided that the tag reacts specifically and stoichiometrically with the cellular constituent in question (2). Today, the repertoire of fluorescent probes is expanding almost daily see Chapter 14). One area that has benefited from the ever-increasing number of fluorescent probes is flow cytometry. 

Organotypic, e.g., mimicking skin or mucosa 3-D from 2-D 3-D/in vivo context, imaging using fluorescent cells possible, biological processes can be assessed (invasion, survival, proliferation, differentiation), antibody staining of fixed matrices possible, 24-weU format Slow (8-day contraction of 3-D matrix), invasion 0-21 days, proliferation not excluded, laborious, cannot be used in HT (31)... 

Fig. 1. A high-throughput platform of the carbohydrate-based microarrays. A high-precision robot designed to produce cDNA microarrays was utilized to spot carbohydrate antigens onto a chemically modified glass slide. The microspotting capacity of this system is approximately 20,000 spots per chip. The antibody-stained slides were then scanned for fluorescent signals with a Biochip Scanner that was developed for cDNA microarrays. The microarray results were subsequently confirmed by at least one of the conventional alternative assays.

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. 

---