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nk, not known. SLE, Systemic lupus erythematosus; RA, rheumatoid arthritis; SS, Sjogren's syndrome; MCTD, mixed connective tissue disease, PSS, progressive systemic sclerosis; CREST, CREST syndrome (variant of scleroderma); PM-DM, Polymyositis-dermatomyositis.

nk, not known. SLE, Systemic lupus erythematosus; RA, rheumatoid arthritis; SS, Sjogren's syndrome; MCTD, mixed connective tissue disease, PSS, progressive systemic sclerosis; CREST, CREST syndrome (variant of scleroderma); PM-DM, Polymyositis-dermatomyositis.

A multitude of assays have been developed to detect the presence of antibodies to DNA. Currently, the most commonly used assays are the IFT on Cri-thidia luciliae, radioimmunoassays (RIAs) such as Farr assay and PEG assay, and enzyme-linked immunosorbent assays (ELISAs). These methods can either be obtained in kit form or be employed as in-house assays. The hemoflagellates Crithidia luciliae which are used in the IFT contain a giant mitochondrion, the kinetoplast, which consists of only double-stranded DNA. To facilitate discrimination between nuclei and kinetoplasts, the slides are counterstained by propidium iodide, which intercalates in the dsDNA helices. Fluorescence of kinetoplasts is taken as an indication of the presence of anti-DNA. After 20 years, this method still is one of the most frequently employed techniques for the detection of anti-DNA. The method is specific and quite sensitive. In RIAs the choice of antigen again is of great importance. The DNA employed has to be bigger than 105 but smaller than 107kDa. Furthermore, the DNA must be double stranded and, to allow quantitation of antibody reactivity, monodisperse in size. This indicates that circular double-stranded bacteriophage DNA (such as from PM2) or plasmids (such as pUC9) are to be preferred.

In ELISA systems, DNA has to be coated to plastic. ssDNA can easily be coated directly, but dsDNA is mostly coated via intermediates such as poly-i-lysine, protamine or methylated BSA. Such precoats introduce problems related to the binding of immune complexes and/or immunoglobulins not directed against DNA to the plates (via the intermediate molecule). An alternative is to make use of biotinyl-ated DNA and coat this via streptavidin to the plates.

Different assay systems are not always comparable, for the following reasons:

1. The source of antigen differs: DNA may be from eukaryotic or prokaryotic origin, be double stranded or single stranded, be polydisperse in size or homogenous, etc.

2. Presentation of the antigen to the antibody differs: in RIAs it is generally in solution, in ELISAs it is coated to plastic; in the Crithidia test DNA is mostly presented intact in cells.

3. Reaction conditions are different: e.g. due to the employed ammonium sulfate precipitation step used in the Farr assay, anti-dsDNA of low avidity is missed with this method; in second antibody techniques such as IFT and ELISA the choice of conjugated antibody is of importance; often, only IgG anti-DNA is measured with these techniques.

General comparison of four different assays using sera of patients with defined SLE leads to high levels of correlations between the various assays (Table 4). However, upon routine screening of sera from patients suspected as having generalized autoimmune diseases, large discrepancies between the different assays are also seen: out of 16 possible reactivity combinations, 11 actually occurred. Sensitivity differences can only partly explain the discrepancies observed between the assays. An important cause is to be found in the avidity of the antibodies (Figure 4). Furthermore, histones or nucleosomes complexed to antinucleosome antibodies may also cause a positive reaction in the Farr assay. It is advisable to quan-titate anti-DNA levels and express these in International Units, to allow comparison between different studies. A World Health Organization (WHO) standard serum to serve this purpose is available from the Central Laboratory of the Blood Transfusion Service (CLB) in Amsterdam. The CLB is the custodian of WHO standards for rheumatology.

Of the methods described, the ELISA is the most

Table 4 Comparison of the disease specificity of four anti-dsDNA assays

Disease Number Percentage of sera positive in

Farr assay Crithidia PEG assay ELISA

Table 4 Comparison of the disease specificity of four anti-dsDNA assays

Disease Number Percentage of sera positive in

Farr assay Crithidia PEG assay ELISA

Active SLE

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