DNA antibodies - Sample
Serum
Specimen stable at 2-8°C for 1 week.

DNA antibodies - Methods
The assays of antibodies to DNA require different approaches in comparison to assays measuring antibodies to proteins because of the physico-chemical properties of DNA and the heterogeneity in its structure. Because antibodies to native or dsDNA have the highest utility in diagnosis, assays highly specific for these DNA forms have been developed.

DNA antibodies - Indirect immunofluorescence assay
See Antinuclear Antibodies – Indirect immunofluorescence assay.

DNA antibodies - Radioimmunoasay
See Appendix.

DNA antibodies - Farr binding
In this assay, a serum is incubated with radiolabelled dsDNA. Plasmid DNA in the form of a closed circle often is used in this assay because of its purity as a dsDNA source. The precipitation of DNA by ammonium sulfate is indicative of the formation of immune complexes and the presence of antibodies (96). Results are reported in terms of the amount or percentage of radiolabelled DNA precipitated. The Farr assay is believed to detect preferentially high avidity antibodies which are associated with renal exacerbations in patients with SLE. An explanation therefore could be that histones containing immune complexes are responsible for a large part of the Farr reactivity in active SLE, and are therefore indirectly implicated in the pathogenesis of lupus nephritis (97).

DNA antibodies - Filter binding assay
This assay resembles the Farr assay in that it involves the interaction of serum antibody with soluble radiolabelled DNA to form an immune complex. This complex is detected by its retention on a nitrocellulose filter, however, thereby eliminating the high salt conditions of the Farr assay. Results are reported in terms of the amount of DNA bound (98, 99)

DNA antibodies - Crithidia luciliae immunofluorescence assay
The hemoflagellata Crithidia luciliae are unicellular organisms that contain a piece of uniform dsDNA in their kinetoplasts (large mitochondria), organelles detectable between the nucleus and the base of the flagellum. Immunofluorescence is used to detect the antibodies binding to dsDNA (100). Results are reported as titer.

DNA antibodies - ELISA
This assay with DNA is not as straightforward as with protein antigens because of the need to assure dsDNA conformation. In addition, dsDNA binds poorly to many plastics, necessitating another component, such as a charged protein, to improve DNA adherence (101).
These assays differ markedly in the types of DNA antigen used - e.g., mammalian versus non-mammalian DNA, the physical chemical form of the DNA- e.g., soluble versus surface-bound, circular versus linear and the conditions for the interaction of antibody with DNA antigen. Whereas the Farr assay demands stable antibody binding in a high salt condition and therefore measures high-affinity antibody, an ELISA detects low- as well as high-affinity antibody. Because of these technical factors, a given serum may show discrepant results when screened by the various assays. Nevertheless, all different assays may be used to assess disease activity because sera taken from an individual patient at various time in disease show consistent behavior in each assay (101, 102). Previously, it has been reported that the presence of antibodies against dsDNA of the IgA class may define a subset of SLE patients suffering from nephritis and arthritis (103). Whether these IgA anti-dsDNA antibodies are a prognostic parameter in patients with SLE needs to be further investigated.

DNA antibodies - Turbidimetry
Turbidimetric and Nephelometric Assays are convenient techniques for measuring the rate of formation of immune complexes in solution. Nephelometry is a technique of determining the concentration of solids suspended in a liquid or a gas by use of a nephelometer. Turbidimetry is a measurement of the turbidity (cloudiness) of a solution or suspension in which the amount of transmitted light is quantified with a spectrophotometer or estimated by visual comparison with solutions of known turbidity. Nephelometry measures the amount of light scattered or deflected from its path by macromolecules in solution. Turbidometry measures the reflection of transmitted light The technique utilizes an incidental light beam that passes through a test solution in a given direction. In dilute solution antigens and antibodies form complexes that scatter light. If the amount of antibody is kept constant, the number of complexes varies in direct proportion to the antigen concentration. The amount of scattered light or the reduction of transmitted light of an unknown sample can therefore be compared with the signal from dilutions of a reference serum of known antigen concentration, and the antigen concentration present in the unknown sample can then be determined.The method can be used to measure most serum proteins including antibodies.In this assay partially purified antigen is mixed with the test serum resulting in the formation of precipitating antibody-antigen complexes. The complexes are measured by the increased scattering of light as they are formed.

    Indirect immunofluorescence assay
    Radioimmunoasay
    Farr binding
    Filter binding assay
    Crithidia luciliae immunofluorescence assay
    ELISA
    Turbidimetry (nephelometry)

Material Pattern:ds-DNA Substrate: Crithidia luciliae Description n-DNA positive sera show a staining fluorescence in the kinetoplast, showed by the nucleus and basal. Method Indirect immunoflourescence method.