Extractable nuclear antigen

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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. [1]

Contents

Clinical applications

An extractable nuclear antigen panel, or an ENA panel, tests for presence of autoantibodies in the blood that react with proteins in the cell nucleus. It is usually done as a follow-up to a positive antinuclear antibody (ANA) test and when one is showing symptoms of an autoimmune disorder. The ANA tests for the presence or absence of autoantibodies, while the ENA panel evaluates which proteins in the cell nucleus the autoantibodies recognize. The ENA panel helps diagnosis, distinguish between, and monitor the progression of autoimmune diseases and is performed with a simple blood draw. While the levels of autoantibodies may fluctuate through one's life, once one develops autoantibodies, one will always have them. Autoantibodies to these antigens are associated with particular connective tissue disorders. Indeed, in 84.3% of positive anti-ENA samples, ANA reagents were also found. [1] The use of anti-ENA autoantibody tests can serve as additional verification of an autoimmune disorder, because a positive ANA test alone does not suffice for diagnosis. In fact, low levels of ANAs can be found in healthy patients. The applications of anti-ENA testing varies from excluding patient groups from specific groups, connective tissue diseases, and to monitor disease activity. In essence, it allows clinicians to exclude specific autoimmune disorders if a particular autoantibody is not present, and allows clinicians to track progression of a disease if the levels of these autoantibodies increase or decrease. To confirm the presence of anti-ENAs, it is currently recommended to use two or more methods to confirm anti-ENAs to avoid false positives. The diagnosis of autoimmune connective tissue diseases (CTDs) is done through analysis of clinical symptoms and signs, but also through the identification of the autoantibodies directed against nuclear antigens. A 2002 paper also seeks to compare the diagnostic tests used in immunology laboratories to measure anti-ENAs and ways to improve this testing and reporting. Double immunodiffusion (DID) and counterimmunoelectrophoresis (CIEP), two forms of gel-based techniques, are used to gain information on the clinical significance and the role of these antibodies in those with CTDs. [2]

Techniques

Gel-based

Since the discovery of ENAs, they have been used as a diagnostic tool in connective tissue disease. Two widely used gel-based techniques were used to identify anti-ENAs and their associations to disease in early work, double immunodiffusion (DID) [3] and counterimmunoelectrophoresis (CIEP). Both of these techniques require the precipitation of antigens for valid results. Depending on the anti-ENA being investigated, one technique may be used over the other. For example, Scl-70 antigen is less negatively charged, which can result in the antigen traveling in the same direction as the antibody. This would result in the antibody-antigen complex not precipitating; leading to invalid results. [4] In addition, some anti-SS-B antibodies commonly identified in Sjögren syndrome may not be detected with this method. However, this method is economically feasible and specific to confirm a diagnosis. There are two sensitivities to note when viewing data from these gel-based techniques, assay sensitivity and disease sensitivity. Assay sensitivity is the ability to recognize when an antibody is present, while disease sensitivity is the ability to recognize the frequency in which the antibody occurs in a disease. Due to limitations of gel-based techniques in disease sensitivity, other techniques have been explored in order to increase assay sensitivity without decreasing disease sensitivity. For example, in patients with Systemic lupus erythematous (SLE), only 8–40% have detectable anti-SM when using gel-based assays. CIEP has been shown to be more sensitive than DID. [5]

Hemagglutination, ELISA, Western Blot

Three additional techniques, passive hemagglutination, enzyme linked immunosorbent assay (ELISA), and western blotting (WB), can be used in order to identify ENAs and link them to specific diseases. Passive hemagglutination was popular in the late 1970s, but very few studies have been done using them and was restricted to anti-Sm and anti-ribonuclear protein (RNP) antibodies. [5] Enzyme linked immunosorbent assay (ELISA) has become the most widely used technique for testing for anti-ENAs due to them being simple to perform, quantitative, and high volume output. While this method has increased assay sensitivity and is efficient for high volume labs, they have a much lower disease specificity than alternative techniques. This is due to the inability to properly isolate the ENAs without large costs due to their association with complexes in the nucleus of the cell. Another worry with the ELISA technique is that anti-Sm antibodies have been reported in patients without SLE which would lead to over-investigating, but could be due to the quality of the antigen source used. [5] Western blotting has a major disadvantage in that antibodies targeted against conformational epitopes can not be detected. On top of that, false positive can occur and the disease specificity is lower than other techniques. While each technique has their advantages and disadvantages, ELISA has the least severe disadvantages of potential for false positives (which are less dangerous than false negatives) and expensive. [5]

Many labs use a combination of both of these techniques to improve efficiency without sacrificing specificity. The current recommendation by European Consensus workshops is to screen for positive anti-ENAs with the ELISA technique. A more specific test such as CIEP will follow with samples that are identified as positive. [6]

The six main antigens used in immunological laboratories for detection are Ro, La, Sm, RNP, Scl-70 and Jo1, [7] which are screened for by Ouchterlony double immuno diffusion techniques and confirmed by immunoblotting. On anti-nuclear antibody tests, these antigens have a speckled pattern. [8]

Terminology

ENAs originally referred to proteins found in a saline extract of cell nuclei. [9] Components have since been more clearly identified and in fact include many cytoplasmic molecules. The misnomer, however, has remained. These proteins are intimately associated with various RNA molecules and are thus called ribonucleoproteins, but the nomenclature used for them is often a source of confusion, Sm, Ro and La were named after the first 2 letters of the surnames of the patients in whom they were first found. Two proteins associated with Sjögren syndrome were independently described as antigens A and B, but are now known to be identical to Ro and La respectively, i.e. SS-A = Ro and SS-B = La.

ENA (extractable nuclear antigen) panel tests, test for autoantibodies to proteins in the cell nucleus. The term "extractable" is derived from the ability to remove the autoantibodies from the nuclei with saline and common proteins. The method of identifying these specimens is why they are also referred to as antibodies to saline-extracted antigens. [9]

ENA

Anti-ENA is a grouping of antibodies often used to screen for mixed connective tissue disease (MCTD), Sjögren's syndrome and systemic lupus erythematosus and commonly is composed of six tests: [10]

Sensitivity and specificity of these tests depends on the type of assay employed, and will therefore vary by lab. The following table illustrates the sensitivity and specificity of ENA antibodies at detecting SLE with the ELISA technique.

Antibody (tested using ELISA) Sensitivity (%) for SLE Specificity (%) for SLE
Anti-Ro (SS-A)6180–93
Anti-La (SS-B)27–3588–97
Anti-Sm34–4588–100
Anti-RNP39–6484–97
Reference for all values: [11]

In addition, the use of ENA testing has also been used for the study of wheat related disorders such as celiac disease. A study conducted in 2018 screened patients with wheat related disorders for 10 anti-ENA antibodies.

73% of celiac disease subjects tested positive for anti-histone and was the most prevalent, which is typically associated with drug-induced lupus erythematosus. This implicates a high probability of an autoimmune disorder in patients with wheat-related disorders. [12]

Related Research Articles

<span class="mw-page-title-main">ELISA</span> Method to detect an antigen using an antibody and enzyme

The enzyme-linked immunosorbent assay (ELISA) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand in a liquid sample using antibodies directed against the protein to be measured. ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries.

<span class="mw-page-title-main">Antinuclear antibody</span> Autoantibody that binds to contents of the cell nucleus

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.

Serology is the scientific study of serum and other body fluids. In practice, the term usually refers to the diagnostic identification of antibodies in the serum. Such antibodies are typically formed in response to an infection, against other foreign proteins, or to one's own proteins. In either case, the procedure is simple.

<span class="mw-page-title-main">Anti-neutrophil cytoplasmic antibody</span> Group of autoantibodies

Anti-neutrophil cytoplasmic antibodies (ANCAs) are a group of autoantibodies, mainly of the IgG type, against antigens in the cytoplasm of neutrophils and monocytes. They are detected as a blood test in a number of autoimmune disorders, but are particularly associated with systemic vasculitis, so called ANCA-associated vasculitides (AAV).

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.

<span class="mw-page-title-main">Anti-mitochondrial antibody</span>

Anti-mitochondrial antibodies (AMA) are autoantibodies, consisting of immunoglobulins formed against mitochondria, primarily the mitochondria in cells of the liver.

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.

A latex fixation test, also called a latex agglutination assay or test, is an assay used clinically in the identification and typing of many important microorganisms. These tests use the patient's antigen-antibody immune response. This response occurs when the body detects a pathogen and forms an antibody specific to an identified antigen present on the surface of the pathogen.

<span class="mw-page-title-main">Anti–citrullinated protein antibody</span> Autoantibodies

Anti-citrullinated protein antibodies (ACPAs) are autoantibodies that are directed against peptides and proteins that are citrullinated. They are present in the majority of patients with rheumatoid arthritis. Clinically, cyclic citrullinated peptides (CCP) are frequently used to detect these antibodies in patient serum or plasma.

RA33, also known as heterogeneous nuclear ribonucleoprotein A2/B1, is an autoantigen in human systemic autoimmune diseases.

<span class="mw-page-title-main">Anti-dsDNA antibodies</span> Group of anti-nuclear antibodies

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.

Anti-nRNP is a type of antibody.

<span class="mw-page-title-main">Lupus</span> Human autoimmune disease

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.

Detection of autoantibodies against mutated citrullinated vimentin is part of rheumatoid arthritis (RA) diagnostics, especially in sera negative for rheumatoid factor. Anti-MCV antibodies are a member of the ACPA family, a group of the so-called antibodies to citrullinated protein/peptide antigens.

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.

<span class="mw-page-title-main">Anti-SSA/Ro autoantibodies</span> Type of anti-nuclear autoantibodies

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.

Anti-Scl-70 is an anti-topoisomerase antibody-type of anti-nuclear autoantibodies, seen mainly in diffuse systemic scleroderma, but is also seen in 10–18% of cases of the more limited form of systemic scleroderma called CREST syndrome. Anti Scl-70 antibodies are associated with more severe scleroderma disease.

Antithyroid autoantibodies (or simply antithyroid antibodies) are autoantibodies targeted against one or more components on the thyroid. The most clinically relevant anti-thyroid autoantibodies are anti-thyroid peroxidase antibodies (anti-TPO antibodies, TPOAb), thyrotropin receptor antibodies (TRAb) and thyroglobulin antibodies (TgAb). TRAb's are subdivided into activating, blocking and neutral antibodies, depending on their effect on the TSH receptor. Anti-sodium/iodide (Anti–Na+/I−) symporter antibodies are a more recent discovery and their clinical relevance is still unknown. Graves' disease and Hashimoto's thyroiditis are commonly associated with the presence of anti-thyroid autoantibodies. Although there is overlap, anti-TPO antibodies are most commonly associated with Hashimoto's thyroiditis and activating TRAb's are most commonly associated with Graves' disease. Thyroid microsomal antibodies were a group of anti-thyroid antibodies; they were renamed after the identification of their target antigen (TPO).

Anti-VGKC-complex encephalitis are caused by antibodies against the voltage gated potassium channel-complex (VGKC-complex) and are implicated in several autoimmune conditions including limbic encephalitis, epilepsy and neuromyotonia.

<span class="mw-page-title-main">Congenital heart block</span> Medical condition

The congenital heart block (CHB) is the heart block that is diagnosed in fetus or within the first 28 days after birth, some studies also include the diagnosis during early childhood to the definition of CHB. It refers to the disorder in the electrical conduction system within the heart muscle, which leads to the failure in pumping the blood efficiently into the aorta and the pulmonary trunk. The result of CHB can be first, second, or third-degree (complete) atrioventricular block in which no electric signals move from the atrium to the ventricles

References

  1. 1 2 Banhuk, Fernanda Weyand; Pahim, Bruna Corrêa; Jorge, Alex Sandro; Menolli, Rafael Andrade (30 September 2018). "Relationships among Antibodies against Extractable Nuclear Antigens, Antinuclear Antibodies, and Autoimmune Diseases in a Brazilian Public Hospital". Autoimmune Diseases. 2018: 1–8. doi: 10.1155/2018/9856910 . PMC   6186355 . PMID   30364021.
  2. ↑ Phan, Tri Giang; Wong, Richard C. W.; Adelstein, Stephen (2002). "Autoantibodies to Extractable Nuclear Antigens: Making Detection and Interpretation More Meaningful". Clinical and Diagnostic Laboratory Immunology. 9 (1): 1–7. doi:10.1128/CDLI.9.1.1-7.2002. PMC   119916 . PMID   11777822.
  3. ↑ "Abbexa - Antibodies, Proteins, ELISA kits".
  4. ↑ Bunn, C.; Kveder, T. (1996). "Counterimmunoelectrophoresis and immunodiffusion for the detection of antibodies to soluble cellular antigens". In van Venrooij, W. J.; Maini, R. N. (eds.). Manual of Biological Markers of Disease. Springer Netherlands. pp. 25–36. doi:10.1007/978-94-011-1670-1_3. ISBN   978-94-011-1670-1.
  5. 1 2 3 4 Lock, RJ; Unsworth, DJ (March 2001). "Antibodies to extractable nuclear antigens. Has technological drift affected clinical interpretation?". Journal of Clinical Pathology. 54 (3): 187–90. doi:10.1136/jcp.54.3.187. PMC   1731369 . PMID   11253128.
  6. ↑ Yiannaki, E E; Tzioufas, A G; Bachmann, M; Hantoumi, J; Tsikaris, V; Sakarellos-Daitsiotis, M; Sakarellos, C; Moutsopoulos, H M (1998). "The value of synthetic linear epitope analogues of La/SSB for the detection of autoantibodies to La/SSB; specificity, sensitivity and comparison of methods". Clinical and Experimental Immunology. 112 (1): 152–158. doi:10.1046/j.1365-2249.1998.00558.x. PMC   1904932 . PMID   9566804.
  7. ↑ Prince, HE; Hogrefe, WR (1998). "Evaluation of a line immunoblot assay for detection of antibodies recognizing extractable nuclear antigens". Journal of Clinical Laboratory Analysis. 12 (5): 320–4. doi:10.1002/(sici)1098-2825(1998)12:5<320::aid-jcla13>3.0.co;2-x. PMC   6807698 . PMID   9773966.
  8. ↑ "Immunopathology".
  9. 1 2 "Extractable Nuclear Antigen Antibodies (ENA) panel". Lab Tests online. June 21, 2018. Retrieved April 10, 2019.
  10. ↑ ENA test (QUANTA Lite) product sheet. inovadx.com. URL: http://www.inovadx.com/Products/di_pdfs/708555/628555rEnglish.pdf ENA [ permanent dead link ]. Accessed on: November 5, 2007.
  11. ↑ Lock, R; Unsworth, D (2001). "Antibodies to extractable nuclear antigens. Has technological drift affected clinical interpretation?". Journal of Clinical Pathology. 54 (3): 187–190. doi:10.1136/jcp.54.3.187. PMC   1731369 . PMID   11253128.
  12. ↑ Yang, Yuanyuan; Krishna, Karthik; Deshpande, Payal; Ranganathan, Vinodh; Jayaraman, Vasanth; Wang, Tianhao; Bei, Kang; Krishnamurthy, Hari (18 June 2018). "High Frequency of Extractable Nuclear Autoantibodies in Wheat-Related Disorders". Biomarker Insights. 13. doi:10.1177/1177271918782893. PMC   6024268 . PMID   29977112.
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