Antibodies to double-stranded (ds)DNA, Sm, and U1 RNP

INTRODUCTION — Selected antinuclear antibodies (ANAs), including anti-double-stranded deoxyribonucleic acid (dsDNA) and anti-Sm, are highly specific for the diagnosis of systemic lupus erythematosus (SLE). Antibodies directed against the U1 ribonucleoprotein (RNP) complex are markers for mixed connective tissue disease (MCTD) and may be seen in patients with SLE and other disorders. (See "Clinical manifestations and diagnosis of systemic lupus erythematosus in adults" and "Mixed connective tissue disease".)

The sensitivity of anti-dsDNA, anti-Sm, and anti-U1 RNP antibodies for the diagnosis of SLE is relatively low, but the specificity of anti-dsDNA and anti-Sm antibodies for SLE makes them invaluable tools to assist in the diagnosis of this disease.

This topic will review the clinical significance of anti-dsDNA, anti-Sm, and anti-U1 RNP autoantibodies. The clinical significance of antiribosomal P autoantibodies, which are also highly specific for the diagnosis of SLE, and of other antinuclear antibodies is discussed separately. (See "Antiribosomal P protein antibodies" and "Measurement and clinical significance of antinuclear antibodies".)

ANTI-DNA ANTIBODIES — Antibodies to deoxyribonucleic acid (DNA) can be primarily divided into two groups: those reactive with denatured, single-stranded (ss)DNA and those recognizing native, double-stranded (ds)DNA [1]. Autoantibodies to DNA were first described in the 1950s. Their binding to DNA in cell nuclei, using a Hep-2 cell line substrate, for example, may be visualized by indirect immunofluorescent (IIF) staining (picture 1). (See 'Anti-ssDNA antibodies' below and 'Anti-dsDNA antibodies' below.)

Anti-ssDNA antibodies — Anti-ssDNA antibodies are generally not useful in the diagnosis and management of systemic lupus erythematosus (SLE). Antibodies that identify ssDNA react primarily with the purine and pyrimidine bases that are buried within the beta helix of double-stranded deoxyribonucleic acid (dsDNA) [2]. Because these epitopes are "hidden" when DNA is in its native configuration, antibodies directed against ssDNA do not usually crossreact with dsDNA. Antibodies to ssDNA have the following general properties:

●They are much less specific for SLE than antibodies to dsDNA. As an example, anti-ssDNA antibodies have been reported in rheumatoid arthritis, in drug-related lupus, in healthy relatives of patients with SLE [3], and, less commonly, in other rheumatic diseases and healthy people [2].

●Anti-ssDNA antibody testing is not useful for the management of patients with SLE because the presence and titer of anti-ssDNA antibodies do not correlate well with SLE disease activity.

Anti-dsDNA antibodies — Anti-dsDNA antibodies are useful in the evaluation and management of patients with SLE because of their high specificity for the diagnosis of SLE and because of their association, in some patients, with disease activity. They have received a significant amount of attention for several reasons:

●Anti-dsDNA antibodies are specific for SLE, making the presence of these antibodies very useful for distinguishing patients with SLE from patients with other systemic autoimmune diseases [4]. Anti-dsDNA antibodies are rarely found in patients with other disorders, including rheumatoid arthritis, Sjögren's disease, scleroderma, Raynaud phenomenon, mixed connective tissue disease (MCTD), discoid lupus, myositis, uveitis, juvenile arthritis, antiphospholipid syndrome, Grave's disease, Alzheimer disease, and autoimmune hepatitis [4,5].

●Titers of anti-dsDNA antibodies often fluctuate with SLE disease activity. When anti-dsDNA antibody levels are integrated with other measures of disease activity, they are useful in the clinical management of SLE patients [4,6-8].

●There is a well-recognized association between the presence of high titer immunoglobulin G (IgG) anti-dsDNA antibodies and active glomerulonephritis [2,7-13]. There is also evidence of deposition of dsDNA-containing immune complexes in the glomeruli of patients with active lupus nephritis. These observations have led investigators to believe that anti-dsDNA antibodies are of primary importance in the pathogenesis of lupus nephritis [8,14]. (See "Lupus nephritis: Diagnosis and classification".)

●Although patients with drug-induced lupus typically develop antibodies directed against ssDNA and histone proteins, anti-dsDNA antibodies have been reported in patients treated with minocycline, etanercept, infliximab, and penicillamine. Along with the production of anti-dsDNA antibodies, these patients may develop a syndrome of arthritis, arthralgias, cutaneous vasculitis, and serositis. (See "Drug-induced lupus".)

Measurement of anti-dsDNA — There are four principal methods used to detect and quantify anti-dsDNA antibodies:

●Farr assay – The Farr assay is based upon the precipitation of radioactively labeled DNA-anti-DNA antibody complexes in a 50 percent saturated ammonium sulfate solution. This assay primarily detects high-affinity antibodies to dsDNA [15]. Approximately 50 to 80 percent of all SLE patients have elevated levels of anti-dsDNA antibodies measured by this method; the antibody level appears to correlate closely with disease activity, especially with active proliferative nephritis [2,15]. The Farr assay is no longer widely used in the United States for the detection of anti-dsDNA antibodies because it requires the use of a radioactive DNA as target antigen and because of the expense associated with disposal of radioactive material.

●Crithidia luciliae assay – The Crithidia luciliae assay is an IIF assay that takes advantage of the fact that the basal body of this unicellular hemoflagellate contains a kinetoplast or a giant mitochondrion. The mitochondrion contains a small circle of DNA that consists exclusively of dsDNA [16]. As in the IIF test for antinuclear antibodies (ANAs) using the HEp-2 cell substrate, a semiquantitative measure of anti-dsDNA antibodies is determined by testing serial dilutions of serum on the hemoflagellate substrate [17,18]. The highest titer of serum that stains the kinetoplast is considered the titer of anti-dsDNA antibody in the patient serum. The Crithidia luciliae assay is of comparable sensitivity to the Farr assay.

●ELISA – Enzyme-linked immunosorbent assay (ELISA) is a third method used to detect anti-dsDNA antibodies [19,20]. In this method, the wells of a microtiter plate are coated with dsDNA, which serves as substrate to detect anti-dsDNA antibodies. Human antibodies are detected using an enzyme-conjugated secondary antiserum directed against human immunoglobulin. The enzyme catalyzes a chemical reaction that can be used to quantify the amount of anti-dsDNA antibodies. The dsDNA ELISA test is positive in approximately 70 to 80 percent of patients with SLE. The IgG antibody levels correlate moderately well with active nephritis, and there is a good correlation with disease activity in general. The specificity of a positive test by ELISA for the diagnosis of SLE is approximately 70 percent [21].

●Fluorescent beads – A fourth method to detect anti-dsDNA antibodies involves the use of dsDNA coupled to fluorescent microspheres. The microspheres are incubated with patient serum, and subsequently with phycoerythrin-conjugated secondary antiserum directed against human immunoglobulin. Flow cytometry is used to detect and quantify the level of fluorescence bound to the microspheres, and the level of fluorescence is related to the concentration in the serum [22].

The sensitivity and specificity of each anti-dsDNA antibody detection method for the diagnosis of SLE depend upon the quality of DNA substrate. The assay may detect antibodies directed against ssDNA, as well as dsDNA, if the dsDNA is partially denatured. The resulting assay may have increased sensitivity but decreased specificity for the diagnosis of SLE. In one study of 158 ANA-positive sera, the sensitivity and specificity of an ELISA for anti-dsDNA antibodies for the diagnosis of SLE were 79 and 73 percent. In contrast, the Crithidia luciliae assay was 41 percent sensitive and 99 percent specific for the diagnosis of SLE [21]. Because a positive test for anti-dsDNA antibodies using the Crithidia luciliae assay has high specificity for the diagnosis of SLE, this assay may be preferred for the initial diagnosis of SLE. Because the other assays provide more quantitative results, Farr, ELISA, and fluorescent bead assays may be preferable for monitoring disease activity.

Occasionally, a patient will be unexpectedly found to have a positive test for anti-dsDNA antibodies but a negative ANA test. HEp-2 cells contain abundant dsDNA, and a positive test for anti-dsDNA antibodies should also result in a positive test for ANAs by IIF. However, because anti-dsDNA antibodies may be detected by a variety of assay techniques (as described above), it is possible that the anti-dsDNA assay used, such as the Farr, ELISA, or fluorescent bead assay, may be more sensitive than IIF on HEp-2 cell substrate for the detection of anti-dsDNA antibodies. The clinical significance of anti-dsDNA antibodies detected by one of these assays, in the setting of a negative test for ANA by IIF, is uncertain.

A false-positive anti-dsDNA may be suspected when anti-dsDNA antibodies are detected using the Crithidia luciliae assay, despite a negative result using the IIF assay for ANAs. This can occur because complexes of low-density lipoprotein and IgG may bind nonspecifically to the flagellate basal body [23]. This false-positive test result using the Crithidia substrate may "disappear" if serum is obtained from the patient after overnight fasting.

An ANA-negative and anti-dsDNA antibody-positive result is not possible if the clinical laboratory uses antigen-coated fluorescent microspheres as a screening test for ANAs; the dsDNA-coated microsphere is one of the panel of antigen-coated microspheres used to test for autoantibodies in such assays. Thus, the fluorescent bead ANA screening test would, by definition, be considered positive if anti-dsDNA antibodies are detected. It should be noted that the American College of Rheumatology recommends that solid-phase assays should not be used as the initial test to detect ANAs, as is outlined in their position statement.

Because anti-dsDNA antibody tests have different sensitivities and specificities and because of the possibility of false positive results, it is important for clinicians to know which method is used by their clinical laboratory for the detection of anti-dsDNA antibodies.

Titer, pathogenicity, and disease activity — It is not clear why some patients with anti-dsDNA antibodies experience SLE flares while others do not. In the case of SLE glomerulonephritis, a number of properties of anti-dsDNA antibodies, including avidity for antigen, isoelectric point, isotype, ability to fix complement, and idiotype, may affect their pathogenicity. The location of immune complex deposition within the kidney and the amount of IgG anti-dsDNA antibody deposition may affect the degree of renal damage [24-26]. Some studies suggest that anti-dsDNA antibodies may crossreact with antigens present in the glomerulus and that these cross-reacting antibodies may contribute to disease pathogenesis.

These antibody properties may vary from individual to individual, over time in the same individual, or with disease activity, even in the face of a stable antibody titer. Thus, there are some patients with SLE who have persistently elevated anti-dsDNA antibody measurements despite improvement in disease activity. Similarly, a small but significant minority of patients have active nephritis without elevations of anti-dsDNA antibody titer.

Titers (in the case of the Crithidia luciliae assay) or concentrations (in the case of other assays) of anti-dsDNA antibodies may be helpful in the management of some patients with SLE. Anti-dsDNA antibodies tend to increase when disease is active, and there is a strong association between the level of anti-dsDNA antibodies and glomerulonephritis [8,9], particularly in the setting of hypocomplementemia [8,27]. Some studies have reported exceptions to the association between anti-dsDNA antibody levels and disease activity; as an example, some patients have elevated levels of anti-dsDNA antibodies in the setting of inactive or minimally active lupus [28]. Thus, while the correlation between antibody level and disease activity holds for some patients with SLE, there are other patients for whom this assay has limited clinical utility. For example, there is no relationship between antibody titer and disease activity for neuropsychiatric manifestations of SLE [29]. In general, monitoring anti-dsDNA antibodies is most useful for those patients in whom changes in titer correlate with clinical status before or during flares [4,30].

Distinguishing a flare of SLE from intercurrent infectious diseases (eg, secondary to treatment with immunosuppressive agents), toxic effects of drugs, and unrelated diseases may be challenging. An increase in the level of anti-dsDNA antibodies may be helpful in some patients in making these distinctions. Although, as noted above, there is variability among patients, the test for anti-dsDNA antibodies becomes especially helpful when a given patient follows the characteristic pattern of rising anti-dsDNA antibodies and falling complement in the setting of a flare. However, once a particular patient shows a disassociation between anti-dsDNA antibody level and clinical evidence of nephritis, for example, subsequent changes in anti-dsDNA antibody levels are less likely to accurately reflect disease activity. In this setting, therapeutic decisions must be guided by the clinical picture and, perhaps, by other serological findings such as complement levels. (See "Overview of the management and prognosis of systemic lupus erythematosus in adults", section on 'Laboratory evaluation'.)

ANTI-Sm AND ANTI-U1 RNP ANTIBODIES — The anti-Sm and anti-U1 ribonucleoprotein (RNP) antigen-antibody systems are considered together because the antibodies frequently coexist in patients with systemic lupus erythematosus (SLE). In addition, the Sm and U1 RNP autoantigens co-localize in distinct cellular structures known as small nuclear ribonucleoprotein particles (snRNPs). Autoantibodies directed against Sm and U1 RNP antigens also produce similar (coarse nuclear speckled) staining patterns when detected by indirect immunofluorescent (IIF) using the HEp-2 cell substrate (picture 2).

Sm and U1 RNP antigens, together with the autoantigens Ro and La, are sometimes referred to as "extractable nuclear antigens." This nomenclature is a remnant from early studies in which antigens were removed or "extracted" from a cellular substrate in a saline solution. The extracted antigens could then be used in Ouchterlony immunodiffusion or counterimmunoelectrophoresis assays to detect autoantibodies. Although agarose gel-based assays are rarely used to detect autoantibodies, the term "extractable nuclear antigens" continues to be frequently used when referring to Sm, U1 RNP, Ro, and La antigens.

Anti-Sm antibodies — The Sm antigens are nuclear, non-histone proteins that were initially characterized in 1966 by Eng Tan and were the first nuclear protein autoantigens to be described in SLE [31]. The unusual name of this autoantibody-antigen system ("Sm") reflects the tradition of naming antigens after the person (Stephanie Smith) whose serum was initially used to identify and characterize the proteins [32].

Anti-Sm antibodies bind to one or more of a series of Sm proteins designated SmB, SmD1, SmD2, SmD3, SmE, SmF, and SmG. The Sm proteins comprise a heptameric ring that forms a complex with small, uridine (U)-rich nuclear ribonucleic acids (RNAs) designated U1, U2, U4, and U5. The Sm proteins bind the U RNAs in the cytoplasm and are critical for the import of snRNPs into the nucleus [33]. snRNPs have an important role in processing ("splicing") precursor messenger (m)RNAs into mature mRNAs [14].

Anti-U1 RNP antibodies — Anti-U1 RNP antibodies react with one or more of three proteins (70-kD, A, and C) that are specifically present in the U1 snRNP complex. Unlike the Sm antigens (which bind to U-rich RNAs U1, U2, U4, and U5), 70-kD, A, and C are only present in the U1 snRNP complex. Anti-U1 RNP antibodies are present in a subset of patients with SLE, and high titer anti-U1 RNP antibodies are present in all patients with mixed connective tissue disease, a disorder that is closely related to SLE. Anti-U1 RNP antibodies may also be present in lower titers in other autoimmune diseases, including rheumatoid arthritis, systemic sclerosis, Sjögren's disease, and polymyositis [34].

Methods of measurement — Historically, anti-Sm and anti-U1 RNP antibodies were detected by immunodiffusion or counterimmunoelectrophoresis in agarose gels. These methods are relatively insensitive for the detection of autoantibodies, and it is difficult to use immunodiffusion to quantify antibody levels [31,35]. Most clinical laboratories use "solid-phase assays" to detect these autoantibodies. Solid-phase assays include the enzyme-linked immunosorbent assay (ELISA) and flow cytometry-based assays described for the detection of anti-double-stranded deoxyribonucleic acid (dsDNA) antibodies above [36]. (See 'Anti-Sm and anti-U1 RNP antibodies' above and 'Measurement of anti-dsDNA' above.)

Clinical utility — Anti-Sm antibodies are insensitive but highly specific markers for SLE [37-39]; anti-U1 RNP antibodies lack this specificity [34,39].

Anti-Sm antibodies may be detected in 10 to 50 percent of SLE patients, and estimates of specificity for SLE range from 55 to 100 percent [37-39]. Anti-Sm antibodies generally remain positive, even when a patient has entered remission. In contrast, the titer of anti-dsDNA antibodies may fall into the normal range when a patient's disease is quiescent. The detection of anti-Sm antibodies may, therefore, be especially useful diagnostically when a SLE patient's disease is relatively inactive.

A study of more than 2300 SLE patients examined the clinical associations of anti-Sm antibodies [40]. Anti-Sm antibodies were present in 579 of the 2322 (24.9 percent) patients in the "PROFILE" cohort. Patients with anti-Sm antibodies were more likely to be diagnosed at a younger age and had shorter disease duration at the time of diagnosis than anti-Sm antibody-negative patients. Renal disease was more common in anti-Sm antibody-positive, compared with anti-Sm antibody-negative, SLE patients (43.9 versus 31 percent, p<.001). There was also an association between the presence of anti-Sm antibodies and neuropsychiatric illness, vasculitis, and the presence of anti-dsDNA antibodies.

The presence of high titer anti-Sm antibodies and low complement levels may identify SLE patients who are risk for having "silent" lupus nephritis [41]. Silent lupus nephritis was defined in this study as the presence of histological evidence of SLE-related renal disease in patients who do not have an abnormal urinalysis or renal impairment. High titer anti-Sm antibodies and low complement levels may indicate that a patient may benefit from early kidney biopsy or at least close monitoring for the development of renal dysfunction. (See "Lupus nephritis: Diagnosis and classification", section on 'Clinical features'.)

Anti-U1 RNP antibodies may be found in 3 to 69 percent of patients with SLE [34,39]. Anti-U1 RNP antibodies are always present in patients with mixed connective tissue disease (MCTD) because the presence of these antibodies is part of the definition of this disorder. High levels of anti-U1 RNP antibodies are often detected in patients with MCTD. (See "Mixed connective tissue disease".)

The wide range of sensitivity and specificity of anti-Sm and anti-U1 RNP antibodies for the diagnosis of SLE reflects the variations in methods used to detect these antibodies. There is no single, standardized technique for producing and purifying Sm and U1 RNP antigens. Some assay kits use antigens that have been purified from cells in culture. Other assay kits may use recombinant antigens that have been prepared in prokaryotic or eukaryotic cells and have then been purified. The presence of contaminating proteins in these preparations may greatly affect the performance of the assays. This is especially true because SLE patients frequently have high levels of antibodies directed against cellular and bacterial proteins. Serum antibodies directed against impurities in the autoantigen preparations may produce a false-positive test.

There is also no consensus as to which autoantigens should be included in assay kits. For example, some commercial kits use only the U1 RNP 70-kD protein as the target for measuring anti-U1 RNP antibodies; other companies include all three of the U1 RNP autoantigens (70-kD, A, and C). In a patient who only has anti-U1 RNP antibodies directed against the A protein, testing only for antibodies against the 70-kD protein will produce a false-negative result.

Because the preparation and purification of autoantigens are considered proprietary information, it is difficult for clinical immunology laboratories to predict and determine which commercial assay will perform the best in clinical practice. The limitations of tests for anti-Sm and anti-U1 RNP autoantibodies using solid-phase assays must be kept in mind when evaluating a patient who may have SLE.

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Antinuclear antibodies".)

INFORMATION FOR PATIENTS — UpToDate offers two types of patient education materials, "The Basics" and "Beyond the Basics." The Basics patient education pieces are written in plain language, at the 5^th to 6^th grade reading level, and they answer the four or five key questions a patient might have about a given condition. These articles are best for patients who want a general overview and who prefer short, easy-to-read materials. Beyond the Basics patient education pieces are longer, more sophisticated, and more detailed. These articles are written at the 10^th to 12^th grade reading level and are best for patients who want in-depth information and are comfortable with some medical jargon.

Here are the patient education articles that are relevant to this topic. We encourage you to print or e-mail these topics to your patients. (You can also locate patient education articles on a variety of subjects by searching on "patient info" and the keyword(s) of interest.)

●Beyond the Basics topics (see "Patient education: Antinuclear antibodies (ANA) (Beyond the Basics)" and "Patient education: Systemic lupus erythematosus (Beyond the Basics)")

SUMMARY AND RECOMMENDATIONS

●Antibodies to deoxyribonucleic acid (DNA) can be primarily divided into two groups: those reactive with denatured, single-stranded (ss)DNA and those recognizing native, double-stranded (ds)DNA. Anti-ssDNA antibodies, which react primarily with the purine and pyrimidine bases, are generally not useful in the diagnosis and management of systemic lupus erythematosus (SLE) due to their low specificity for SLE and poor correlation with SLE disease activity. (See 'Anti-DNA antibodies' above and 'Anti-ssDNA antibodies' above.)

●Anti-dsDNA antibodies are useful in the evaluation and management of patients with SLE because of their relatively high specificity for SLE, tendency to fluctuate with disease activity, and association with active glomerulonephritis. Methods for detection of anti-dsDNA include the Farr, Crithidia luciliae, enzyme-linked immunosorbent (ELISA), and fluorescent microsphere assays. The sensitivity and specificity of each method for the diagnosis of SLE depend, in part, upon the quality of DNA substrate. (See 'Anti-dsDNA antibodies' above and 'Measurement of anti-dsDNA' above.)

●A number of properties of anti-dsDNA antibodies may affect their pathogenicity in the kidney, including avidity, isoelectric point, isotype, ability to fix complement, and idiotype. In addition, the location and amount of immune complex deposition in the kidney may affect disease pathogenesis. Testing for anti-dsDNA antibodies may be especially helpful in patients who exhibit the characteristic pattern of rising anti-dsDNA antibodies and falling complement in the setting of a flare with evidence of nephritis. However, this association is not present in some patients, for whom changes in anti-dsDNA antibody levels do not reflect disease activity. (See 'Titer, pathogenicity, and disease activity' above.)

●The anti-Sm and anti-U1 ribonucleoprotein (RNP) antigen antibodies frequently coexist in patients with SLE and produce similar (coarse speckled nuclear) staining patterns when detected by indirect immunofluorescence using the HEp-2 cell substrate. The Sm and U1 RNP autoantigens co-localize in distinct cellular structures known as small nuclear ribonucleoprotein particles (snRNPs). (See 'Anti-Sm and anti-U1 RNP antibodies' above.)

●The Sm antigens are nuclear, non-histone proteins which comprise a heptameric ring that forms a complex with small, uridine-rich nuclear ribonucleic acids (RNAs) to facilitate import of snRNPs into the nucleus. Sm proteins have an important role in the processing of precursor messenger (m)RNAs. Most clinical laboratories use solid-phase assays such as ELISA and flow cytometry-based assays to detect these autoantibodies. (See 'Anti-Sm antibodies' above and 'Anti-U1 RNP antibodies' above and 'Methods of measurement' above.)

●Anti-Sm antibodies are relatively insensitive but highly specific markers for SLE and generally remain positive, even when a patient has entered remission. (See 'Clinical utility' above.)

●Anti-U1 RNP antibodies are always present, by definition, in patients with mixed connective tissue disease (MCTD). Anti-U1 RNP antibodies may also be found in some patients with SLE. The wide range of sensitivity and specificity of anti-Sm and anti-U1 RNP antibodies for the diagnosis of SLE reflects the variations in methods used to detect these antibodies. (See 'Clinical utility' above.)