Anti-double stranded DNA (Anti-dsDNA) antibodies are a group of anti-nuclear antibodies (ANA) the target antigen of which is double stranded DNA. Blood tests such as enzyme-linked immunosorbent assay (ELISA) and immunofluorescence are routinely performed to detect anti-dsDNA antibodies in diagnostic laboratories. They are highly diagnostic of systemic lupus erythematosus (SLE) and are implicated in the pathogenesis of lupus nephritis. [1] [2]
The first evidence for antinuclear antibodies arose in 1948 when Hargraves, Richmond and Morton discovered the LE cell. [3] These abnormal cells, which are found in the bone marrow of persons who have SLE are categorised as polymorphonuclear leukocytes with phagocytosed whole nuclei. [4] Subsequently, in 1957, antibodies to dsDNA were the first autoantibodies to be identified in patients with SLE. [5]
Although the exact mechanism of the generation of dsDNA antibodies is still unknown, it is likely that extracellular DNA is one cause of an immune response against dsDNA. There is a great deal of evidence supporting the idea that dead or dying cells are one major source of this extracellular DNA. [6] Apoptosis is the highly organised process of programmed cell death in which the cell degrades the nuclear DNA and signals for phagocytosis. In people with SLE and other autoimmune disorders this process is thought to be defective, causing either an increase in cell death and/or a decrease in the rate of dead cell clearance. [7]
There is a higher rate of apoptosis in people with SLE and various changes in genes and proteins have been implicated in the defects in apoptosis. These include increased levels of soluble Fas and bcl-2 and polymorphisms in the programmed cell death 1 and runt-related transcription factor X1. [7]
Blebs on apoptotic cells contain nearly all the autoantigens found in SLE, and phagocytes bind these apoptotic cells and phagocytose them. If this process is defective, these autoantigens can be released into the circulation allowing an immune response. Serum amyloid P component is a protein that is thought to aid in the clearance of chromatin produced by apoptotic cells and deficiencies in this protein have been shown (in mice) to cause spontaneous formation of ANA. Autoantigens present on the blebs of apoptotic cells are also prone to modification, which can increase their immunogenicity. [7] [8]
Upon release of nuclear proteins and chromatin, antigen presenting cells, such as dendritic cells and macrophages, display these antigens to T helper cells. Although the details of this process are still controversial, evidence shows that to produce an immune response, DNA must activate an antigen presenting cell to produce type 1 interferons. This cytokine serves to induce maturation of plasmacytoid dendritic cells (PDCs) so that they can display their antigens to T helper cells. The mechanism in which eukaryotic DNA activates these cells is still as yet unclear; however, immunogenic CpG sequences have been found to either activate PDCs or act as adjuvant in the response to eukaryotic DNA. CpG motif DNA acts via the pattern recognition receptor, toll-like receptor 9, found highly expressed in PDCs and B cells. The T helper cells then activate B cells, which are also in the presence of these antigens, causing the production of autoantibodies. [6] [9] [10] [11]
Anti-dsDNA antibodies can also be produced through infection via a mechanism known as molecular mimicry. Upon exposure to pneumococcal polysaccharides, cross reactive antibodies between dsDNA and pneumococcal polysaccharides are produced in lupus. [12] Epstein–Barr virus is also known to induce dsDNA antibodies, as seen after immunisation of animals with EBNA-1 epitopes. [13]
Anti-dsDNA antibodies might also be created secondary to the production of antibodies to other proteins within the nucleosome. Mice that have T cells directed towards the nucleosome can elicit a response to other antigens such as dsDNA and histone via a mechanism known as antigen spreading. This effect can also occur after an infection causes the production of autoantibodies to other structures within the nucleus. [13] [14]
Anti-dsDNA antibodies are incredibly specific for SLE, with studies quoting nearly 100%, and are therefore used in the diagnosis of SLE. Higher titres of anti-dsDNA antibodies are more suggestive of SLE and lower titres can be found in people without the disease. In contrast to the high specificity, estimates of 25–85% have been observed for the sensitivity of anti-dsDNA in SLE. Therefore, presence of anti-dsDNA antibodies are suggestive of SLE, however an absence of the antibodies does not rule out the disease. [1]
The levels of circulating anti-dsDNA antibodies fluctuate with disease activity in SLE. Increases in titres of the antibodies can coincide with, or even precede an increase of disease activity. For this reason titres are serially monitored by clinicians to assess disease progression. Titres are monitored more often in cases of more active lupus than that of less active lupus at intervals of 1–3 months and 6–12 months, respectively. [1]
Anti-dsDNA antibodies are highly associated with glomerulonephritis in SLE, although some patients with high titers of anti-dsDNA antibodies do not develop renal disease. This is most likely due to the fact that anti-dsDNA are a heterogeneous population, some of which have been found not to be pathogenic. Anti-dsDNA antibodies can be present in normal individuals, however these antibodies are usually low avidity IgM isotype. In contrast, pathogenic anti-dsDNA antibodies found in SLE are usually of IgG isotype and show high avidity for dsDNA. [15] One possible mechanism for anti-dsDNA and their role in nephritis is the formation of immune complexes that arise by indirect binding to DNA or nucleosomes that are adhered to the glomerular basement membrane (GBM). Another mechanism is direct binding of antibodies to GBM antigens such as C1q, nucleosomal proteins, heparin sulphate or laminin, which can initiate an inflammatory response by activating complement. They can also be internalised by certain molecules on the GBM cells and cause inflammatory cascades, proliferation and alteration of cellular functions. [2] [16] [17]
Patients with rheumatoid arthritis can develop anti-dsDNA antibodies, however they are usually treatment related. Anti-TNFα biological therapies, such as adalimumab, infliximab and etanercept, can often induce the production of anti-dsDNA antibodies. They are usually low avidity and are only detectable transiently after treatment. The presence of these antibodies can induce a lupus-like syndrome in some cases. [18] [19]
Infection with viral pathogens can induce anti-dsDNA antibodies transiently. Human immunodeficiency virus, parvovirus B19 and BK virus are known to induce these antibodies. [20] [21]
There is little evidence supporting the association between anti-dsDNA antibodies and other diseases. Occasionally the monoclonal proteins produced by myeloma patients can be anti-dsDNA. Also, some patients with type 1 autoimmune hepatitis produce anti-dsDNA antibodies. [22] [23]
A variety of assay formats can be used to detect and quantify anti-dsDNA antibodies but there is no 'gold standard' for diagnostic purposes and the concordance between different assays/methods is low. [24]
The Farr assay is used to quantify the amount of anti-dsDNA antibodies in serum. Ammonium sulphate is used to precipitate antigen-antibody complexes that form if the sera contains antibodies to dsDNA. The quantity of these antibodies is determined by using radioactively labelled dsDNA. Although this test is very specific, it is of little use in routine diagnostic laboratories due to its laboriousness and use of radioactive materials. The Farr assay is one of the only tests available that detects high avidity antibodies (along with Crithidia luciliae) and also has the ability to detect antibodies of any isotype. [15]
The polyethylene glycol (PEG) assay precipitates DNA-antibody complexes, similar to the Farr Assay. However, unlike the Farr Assay it does not dissociate the low avidity antibody complexes, allowing for the detection of both high and low avidity anti-dsDNA antibodies. [25]
Animal tissue was the first substrate for immunofluorescent detection of antinuclear antibodies and has been in use since the late 1950s. Liver and kidney tissue sections from animals such as rats are used to identify anti-dsDNA antibodies. This substrate has largely been superseded by the use of HEp-2 cells. [1]
Hep-2 cells, originally of laryngeal carcinoma origin, are actually a contamination of HeLa cells. [26] They are routinely used in the diagnosis of ANA in diagnostic laboratories. HEp-2 cells provide a greater ability to differentiate patterns of ANA than animal sections, due to the large nuclei and high mitotic rate of the cell line. Upon incubation with serum containing anti-dsDNA antibodies and fluorescent labelled secondary antibodies, homogeneous staining of interphase nuclei and condensed chromosomal staining of mitotic cells can be seen. [27]
Crithidia luciliae is a haemoflagellate protist with an organelle known as the kinetoplast. This organelle contains a high concentration of circular DNA with no recognisable nuclear antigens, allowing for the reliable detection of anti-dsDNA antibodies. The kinetoplast fluoresces if serum contains high avidity anti-dsDNA antibodies. This test has a higher specificity than EIA because it uses unprocessed DNA. Processed DNA can contain regions of ssDNA, allowing detection of anti-ssDNA antibodies, which can give false positive results. [1] [28]
EIA (enzyme immunoassay) detects antibodies using a DNA-coated polystyrene microtitre plate. The DNA used in these assays is often recombinant dsDNA or from calf thymus extract. [29] Upon incubation with serum containing anti-dsDNA antibodies, the antibodies will bind to the DNA and can then be visualised using enzyme-linked secondary antibodies. This assay can be quantitative or semi-quantitative, allowing for estimations of the levels of anti-dsDNA antibodies. This test can produce false positives due to contamination of ssDNA from denatured dsDNA. EIA detects low and high avidity anti-dsDNA antibodies, increasing its sensitivity and reducing its specificity. [1]
Flow cytometry for the detection of ANA uses multiplexed polystyrene beads coated with multiple autoantigens, such as SSA, SSB, Sm, RNP, Scl-70, Jo-1, dsDNA, centromere B and histone. Serum is incubated with the beads and in the presence of anti-dsDNA antibodies, or any other ANA, the antibodies will bind and fluorescent labelled secondary antibodies will be used for detection. The beads are run through a flow cell which uses a laser to detect fluorescence. [30] [31]
Similar to the flow cytometry method of ANA detection, the MIA uses wells containing autoantigens and HEp-2 extract coated beads. The bead sets are coated with specific autoantigens and can be detected individually to allow identification of the particular autoantibody. Automated analysis of the well fluorescence allows for rapid detection of autoantibodies. [30] [32]
Microarrays are a newly emerging method for the detection of ANA. Individual autoantigens are deposited in an array of dots onto a surface such as polystyrene. A single array could consist of hundreds of autoantigens for screening of multiple autoimmune diseases simultaneously. If anti-dsDNA antibodies are present, incubation of serum and the microarray allow for binding and the dots can then be visualised using a fluorescent labelled anti-IgG antibody. [33]
As a result of the highly specific nature of antibodies, they can be engineered to target and bind key motifs. These motifs can be key features within the pathogenesis of particular diseases, for example human papillomavirus. [34]
In immunology, autoimmunity is the system of immune responses of an organism against its own healthy cells, tissues and other normal body constituents. Any disease resulting from this type of immune response is termed an "autoimmune disease". Prominent examples include celiac disease, diabetes mellitus type 1, Henoch–Schönlein purpura (HSP), systemic lupus erythematosus (SLE), Sjögren syndrome, eosinophilic granulomatosis with polyangiitis, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, Addison's disease, rheumatoid arthritis (RA), ankylosing spondylitis, polymyositis (PM), dermatomyositis (DM), and multiple sclerosis (MS). Autoimmune diseases are very often treated with steroids.
Antinuclear antibodies are autoantibodies that bind to contents of the cell nucleus. In normal individuals, the immune system produces antibodies to foreign proteins (antigens) but not to human proteins (autoantigens). In some cases, antibodies to human antigens are produced.
An autoantibody is an antibody produced by the immune system that is directed against one or more of the individual's own proteins. Many autoimmune diseases are associated with such antibodies.
Anti-mitochondrial antibodies (AMA) are autoantibodies, consisting of immunoglobulins formed against mitochondria, primarily the mitochondria in cells of the liver.
Extractable nuclear antigens (ENAs) are over 100 different soluble cytoplasmic and nuclear antigens. They are known as "extractable" because they can be removed from cell nuclei using saline and represent six main proteins: Ro, La, Sm, RNP, Scl-70, Jo1. Most ENAs are part of spliceosomes or nucleosomes complexes and are a type of small nuclear ribonucleoprotein (snRNPS). The location in the nucleus and association with spliceosomes or nucleosomes results in these ENAs being associated with additional RNA and proteins such as polymerases. This quality of ENAs often makes it difficult to purify and quantify their presence for clinical use.
Mixed connective tissue disease, commonly abbreviated as MCTD, is an autoimmune disease characterized by the presence of elevated blood levels of a specific autoantibody, now called anti-U1 ribonucleoprotein (RNP) together with a mix of symptoms of systemic lupus erythematosus (SLE), scleroderma, and polymyositis. The idea behind the "mixed" disease is that this specific autoantibody is also present in other autoimmune diseases such as systemic lupus erythematosus, polymyositis, scleroderma, etc. MCTD was characterized as an individual disease in 1972 by Sharp et al., and the term was introduced by Leroy in 1980.
Integrin alpha M (ITGAM) is one protein subunit that forms heterodimeric integrin alpha-M beta-2 (αMβ2) molecule, also known as macrophage-1 antigen (Mac-1) or complement receptor 3 (CR3). ITGAM is also known as CR3A, and cluster of differentiation molecule 11B (CD11B). The second chain of αMβ2 is the common integrin β2 subunit known as CD18, and integrin αMβ2 thus belongs to the β2 subfamily integrins.
Anti-cardiolipin antibodies (ACA) are antibodies often directed against cardiolipin and found in several diseases, including syphilis, antiphospholipid syndrome, livedoid vasculitis, vertebrobasilar insufficiency, Behçet's syndrome, idiopathic spontaneous abortion, and systemic lupus erythematosus (SLE). They are a form of anti-mitochondrial antibody. In SLE, anti-DNA antibodies and anti-cardiolipin antibodies may be present individually or together; the two types of antibodies act independently. This is in contrast to rheumatoid arthritis with systemic sclerosis (scleroderma) because anti-cardiolipin antibodies are present in both conditions, and therefore may tie the two conditions together.
Anti-topoisomerase antibodies (ATA) are autoantibodies directed against topoisomerase and found in several diseases, most importantly scleroderma. Diseases with ATA are autoimmune disease because they react with self-proteins. They are also referred to as anti-DNA topoisomerase I antibody.
Anti-thrombin antibodies are autoantibodies directed against thrombin that may constitute a fraction of lupus anticoagulant and are seen an increased levels in systemic lupus erythematosus.
Interferon alpha-1 is a protein that in humans is encoded by the IFNA1 gene.
Tripartite motif-containing protein 21, also known as E3 ubiquitin-protein ligase TRIM21, is a protein that in humans is encoded by the TRIM21 gene. Alternatively spliced transcript variants for this gene have been described but the full-length nature of only one has been determined. It is expressed in most human tissues.
60 kDa SS-A/Ro ribonucleoprotein is a protein that in humans is encoded by the TROVE2 gene.
Ficolin-3 is a protein that in humans is encoded by the FCN3 gene. Ficolin-3 was initially identified as H-ficolin, in which H is after the Hakata antigen that was previously found as an autoantigen in patients who lived in the city of Hakata.
Lupus erythematosus is a collection of autoimmune diseases in which the human immune system becomes hyperactive and attacks healthy tissues. Symptoms of these diseases can affect many different body systems, including joints, skin, kidneys, blood cells, heart, and lungs. The most common and most severe form is systemic lupus erythematosus.
RA33, also known as heterogeneous nuclear ribonucleoprotein A2/B1, is an autoantigen in human systemic autoimmune diseases.
Lupus, technically known as systemic lupus erythematosus (SLE), is an autoimmune disease in which the body's immune system mistakenly attacks healthy tissue in many parts of the body. Symptoms vary among people and may be mild to severe. Common symptoms include painful and swollen joints, fever, chest pain, hair loss, mouth ulcers, swollen lymph nodes, feeling tired, and a red rash which is most commonly on the face. Often there are periods of illness, called flares, and periods of remission during which there are few symptoms.
Anti-histone antibodies are autoantibodies that are a subset of the anti-nuclear antibody family, which specifically target histone protein subunits or histone complexes. They were first reported by Henry Kunkel, H.R. Holman, and H.R.G. Dreicher in their studies of cellular causes of lupus erythematosus in 1959–60. Today, anti-histone antibodies are still used as a marker for systemic lupus erythematosus, but are also implicated in other autoimmune diseases like Sjögren syndrome, dermatomyositis, or rheumatoid arthritis. Anti-histone antibodies can be used as a marker for drug-induced lupus.
Anti-SSA autoantibodies are a type of anti-nuclear autoantibodies that are associated with many autoimmune diseases, such as systemic lupus erythematosus (SLE), SS/SLE overlap syndrome, subacute cutaneous lupus erythematosus (SCLE), neonatal lupus and primary biliary cirrhosis. They are often present in Sjögren's syndrome (SS). Additionally, Anti-Ro/SSA can be found in other autoimmune diseases such as systemic sclerosis (SSc), polymyositis/dermatomyositis (PM/DM), rheumatoid arthritis (RA), and mixed connective tissue disease (MCTD), and are also associated with heart arrhythmia.
Crithidia luciliae is a flagellate parasite that uses the housefly, Musca domestica, as a host. As part of the family of Trypanosomatidae, it is characterised by the presence of a kinetoplast, a complex network of interlocking circular double-stranded DNA (dsDNA) molecules. The presence of the kinetoplast makes this organism important in the diagnosis of systemic lupus erythamatosus (SLE). By using C. luciliae as a substrate for immunofluorescence, the organelle can be used to detect anti-dsDNA antibodies, a common feature of the disease.