Detection of AntiRoSSA Antibodies

A hallmark of eukaryotic cells is their separation into subcellular compartments. The nucleus contains many internal nuclear domains, including the nuclear envelope, the nucleolus (Carmo-Fonseca et al. 2000), splicing speckles (Spector 1993), and a variety of nuclear bodies (Gall 2000, Zhong et al. 2000). This spatial organization reflects the requirement for spatial and temporal coordination of nuclear processes: Nuclear proteins with related functions, such as DNA replication, transcription of RNA, or subsequent RNA splicing, are assembled in multiprotein/nucleic acid complexes and thus colocalize at the cytological level.

During the past 25 years, characterization of ANAs has helped to identify many nuclear proteins by their subcellular localization. The commonly used technique for detection of ANA, immunofluorescence microscopy, represents a valuable tool for both the clinician to identify certain autoantibody specificities to diagnose subsets of systemic autoimmune diseases and the biomedical researcher to analyze the architecture and function of the cell nucleus. Immunofluorescence enables the visualization of antigens and the identification of structures in cells and tissues. The emitted signal of excited fluorochromes is viewed against a black background, thus providing high contrast. In addition, fluorescence imaging delivers superb specificity. These advantages of immunofluorescence techniques can be further enhanced by confocal laser scanning microscopy. Confocal microscopes differ from conventional (wide-field) microscopes because they fade out out-of-focus signals. In a confocal microscope, most of the out-of-focus light is excluded from the final image, which greatly increases the contrast and the visibility of fine details in the specimen. Thus, confo-cal microscopy may lead to refined analyses of nuclear autoantigens as well as new opportunities for differential diagnosis of systemic rheumatic diseases and identification of (new) autoantigens, which might have been missed by conventional epi-fluorescence microscopy (Hemmerich and von Mikecz 2000).

Figure 24.1 shows confocal imaging of an autoantibody that is specifically directed against Ro52 (Fig. 24.1C, lane 2). Confocal sectioning of a human epidermal cell line (HEp-2) reveals a unique staining pattern of bright speckles embedded in a weak homogeneous staining of the nucleoplasm (Fig. 24.1A, B, green color), whereas nucleoli as well as the cytoplasm are not labeled. The typical feature of a confocal Ro/SSA image is the visibility of one speckle in three to four focal planes (Fig. 24.1A, arrows), suggesting that nucleoplasmic Ro/SSA speckles have a size of approximately 2 |im. Thus, Ro/SSA speckles can be easily and unmistakably distinguished from splicing speckles, which occupy a larger subnuclear area, and from other nuclear structures, such as nucleoli, centromeres, and nuclear bodies; the latter display a staining pattern of smaller dots.

Many clinical laboratories still use immunodiffusion or counterimmunoelectro-phoresis for the detection of anti-Ro/SSA antibodies, whereas biomedical research laboratories apply immunofluorescence, immunoblotting, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) techniques. The early work leading to the discovery of anti-Ro/SSA antibodies was based on immunodiffusion using extracts from human thyroid, spleen, and calf thymus (Clark et al. 1969,Alspaugh and Tan 1975). The method is easy and quick; however, the substrates are rather unspe-cific and thus may lead to misleading results, especially when appropriate control sera are not available. In terms of specificity, immunoblotting and immunprecipitation are the methods of choice. Extracts of MOLT-4 and HeLa cell lines have been used successfully for the detection of Ro/SSA antigens by immunoblotting (Ben-Chetrit et al. 1988, Rader et al. 1989). Immunoblotting enables the exact identification of the target antigens by determination of their molecular weight. With the cloning of Ro/SSA52 and Ro/SSA60 proteins, purified recombinant proteins have become the preferred substrates in ELISAs (Chan et al. 1991). However, ELISAs are expensive and prone to false-positive results, since the technique does not allow definition of molecular and cell biological properties of the substrate.

In contrast, a combination of immunofluorescence and immunoblotting provides information on the subcellular distribution and molecular weight (= integrity) of target antigens, and thus allows exact identification of ANA specificities. Moreover, the use of confocal microscopes delivers subnuclear immunofluorescent staining patterns in such detail that nuclear autoantigens can be distinguished by exclusive employment of this imaging technique (see previously). Since the new instruments are easy to use and become less and less expensive, it is safe to assume that confocal laser scanning microscopy will find its way from the research laboratories to the clinical routine laboratories.

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