AnalyticalA comparison of ELISA assays as routine diagnostic test for detection of autoantibodies against extractable nuclear antigens
Introduction
A common feature of connective tissue diseases is the presence of autoantibodies directed against various nuclear components. Serological characterisation of autoantibodies to nuclear antigens (ANA) is a well-established tool in the diagnosis of these diseases, such as systemic lupus erythematosus (SLE), Sjögren’s syndrome, progressive systemic sclerosis, rheumatoid arthritis, and mixed connective tissue disease (1). Detection of ANA is usually performed by indirect immunofluorescence. These antibodies can be directed against many different antigens. A group of antigens is extractable with a saline solution and called extractable nuclear antigens (ENA): Smith antigen (Sm), ribonuclear protein (RNP), Sjögren syndrome-associated antigen A & B (SS-A and SS-B), Scleroderma-associated antigen (Scl-70) and Jo-1. Identification of distinct ENA antibody profiles can have diagnostic as well as prognostic implications (1).
For detection of ENA autoantibodies, several techniques have been developed. Double immunodiffusion is probably most widely used (2), but this method is limited by speed, rather low assay sensitivity and requires well trained and experienced technicians (3). Counterimmunoelectrophoresis (CIE) has a slightly better sensitivity and a lower turn-around time (4). Immunoblotting (IB) after electrophoresis is a reliable method because it is not limited by impurities of the antigen preparation (6). However, preparing immunoblots is time consuming and requires sophisticated laboratory equipment. The immunoblots are also commercially available (6). During the past several years, enzyme-linked immunosorbent assays (ELISA) have been developed for routine analysis of ENA autoantibodies 7, 8, using biochemically purified antigens or recombinant antigens (3).
We compared three commercially available screening ELISA assays and two typing ELISA assays with a currently used combination of CIE and IB.
Section snippets
Specimens
Sera were diluted 80-fold and screened by indirect immunofluorescence assay on Hep-2 cells (Immunoconcepts, BioMedical Diagnostics, Brugge, Belgium [BMD]) for the presence of ANA. ANA positive samples were subsequently analyzed for the presence of ENA using CIE/IB. Sera from 180 ANA positive samples, selected from a large sample collection to ensure a wide variety of autoantibodies, were evaluated for the presence of antibodies directed against SS-A, SS-B, Sm, and RNP. Besides samples with
Results
The comparison of the screening ELISAs with the CIE/IB method are shown in Table 1. For the Relisa®ENA and ENA-LISA™ Polyvalent method the cases with borderline reactivity (bord.) are also shown. The agreement (considering borderline to be negative) between the CIE/IB and ELISAs was 90%, 95%, and 93% for Relisa®ENA, ENA-LISA™ Polyvalent and The Milenia ENA screen, respectively. The Relisa®ENA ELISA showed a considerable number of positive results (17 cases) and also a number of sera with
Discussion
In recent years, the detection and differentiation of autoantibodies to nuclear antigens has achieved clinical importance, since certain antibody profiles may help to distinguish between various systemic rheumatic diseases. In this analytical evaluation, we compared the performance of ELISAs with that of conventional diagnostic methods currently in use for ANA characterization. We evaluated 57 ANA-positive and ENA-positive sera and 129 ANA-positive and ENA-negative sera for the presence of
Acknowledgements
The test kits used in this study were kindly provided by the manufacturers.
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Sm peptides in differentiation of autoimmune diseases
2011, Advances in Clinical ChemistryCitation Excerpt :The biologic relevance of these findings remains to be determined. Various techniques, in combination with a variety of different antigens, have been proposed for the detection of Sm antibodies: passive hemagglutination [39], counterimmunoelectrophoresis [40–46], double immunodiffusion, immunoblotting [41,42,44–46], immunoprecipitation [44], ELISA [29,47–55], line immunoassays (LIAs) [56–58], protein microarrays ([60], reviewed in Refs. [61,62]), bead arrays [63–66], a filtration-assisted nanodot array luminometric immunoassay (NALIA) [67], and the UltraPlex platform [68]. In addition, a broad spectrum of antigens can be used for the detection of anti-Sm antibodies, namely native antigens from different sources, purified or recombinant proteins, and synthetic peptides (reviewed in Refs. [1,29,40–56]).
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