![DsDNA antibodies](https://upload.wikimedia.org/wikipedia/commons/2/29/DsDNA_antibodies.jpg)
Antinuclear antibodies (ANAs, also known as antinuclear factor or ANF) are
autoantibodies that bind to contents of the
cell nucleus. In normal individuals, the immune system produces antibodies to foreign proteins (
antigen
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune respons ...
s) but not to human proteins (
autoantigen
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". ...
s). In some cases, antibodies to human antigens are produced.
There are many subtypes of ANAs such as
anti-Ro antibodies,
anti-La antibodies,
anti-Sm antibodies,
anti-nRNP antibodies,
anti-Scl-70 antibodies
Anti-Scl-70 (also called anti-topoisomerase I after the type I topoisomerase target) is an anti-topoisomerase antibody-type of anti-nuclear autoantibodies, seen mainly in diffuse systemic scleroderma (with a sensitivity of 28–70%), but is als ...
,
anti-dsDNA 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 p ...
,
anti-histone antibodies 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 i ...
, antibodies to nuclear pore complexes,
anti-centromere antibodies
Anti-centromere antibodies (ACAs; often styled solid, anticentromere) are autoantibodies specific to centromere and kinetochore function. They occur in some autoimmune diseases, frequently in limited systemic scleroderma (formerly called CREST sy ...
and
anti-sp100 antibodies. Each of these antibody subtypes binds to different proteins or protein complexes within the nucleus. They are found in many disorders including
autoimmunity
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". ...
,
cancer
Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
and
infection
An infection is the invasion of tissues by pathogens, their multiplication, and the reaction of host tissues to the infectious agent and the toxins they produce. An infectious disease, also known as a transmissible disease or communicable dis ...
, with different prevalences of antibodies depending on the condition. This allows the use of ANAs in the diagnosis of some autoimmune disorders, including
systemic lupus erythematosus,
Sjögren syndrome,
scleroderma
Scleroderma is a group of autoimmune diseases that may result in changes to the skin, blood vessels, muscles, and internal organs. The disease can be either localized to the skin or involve other organs, as well. Symptoms may include areas ...
,
mixed connective tissue disease
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 ...
,
polymyositis
Polymyositis (PM) is a type of chronic inflammation of the muscles (inflammatory myopathy) related to dermatomyositis and inclusion body myositis. Its name means "inflammation of many muscles" ('' poly-'' + '' myos-'' + '' -itis''). The inflam ...
,
dermatomyositis
Dermatomyositis (DM) is a long-term inflammatory disorder which affects skin and the muscles. Its symptoms are generally a skin rash and worsening muscle weakness over time. These may occur suddenly or develop over months. Other symptoms may inc ...
,
autoimmune hepatitis
Autoimmune hepatitis, formerly known as lupoid hepatitis, plasma cell hepatitis, or autoimmune chronic active hepatitis, is a chronic, autoimmune disease of the liver that occurs when the body's immune system attacks liver cells, causing the live ...
and
drug induced lupus.
The ANA test detects the autoantibodies present in an individual's
blood serum. The common tests used for detecting and quantifying ANAs are
indirect immunofluorescence and
enzyme-linked immunosorbent assay
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 ...
(ELISA). In immunofluorescence, the level of autoantibodies is reported as a
titre
Titer (American English) or titre (British English) is a way of expressing concentration. Titer testing employs serial dilution to obtain approximate quantitative information from an analytical procedure that inherently only evaluates as positiv ...
. This is the highest dilution of the serum at which autoantibodies are still detectable. Positive autoantibody titres at a dilution equal to or greater than 1:160 are usually considered as clinically significant. Positive titres of less than 1:160 are present in up to 20% of the healthy population, especially the elderly. Although positive titres of 1:160 or higher are strongly associated with autoimmune disorders, they are also found in 5% of healthy individuals.
Autoantibody screening is useful in the diagnosis of autoimmune disorders and monitoring levels helps to predict the progression of disease.
A positive ANA test is seldom useful if other clinical or laboratory data supporting a diagnosis are not present.
Immunity and autoimmunity
The human body has many defense mechanisms against
pathogen
In biology, a pathogen ( el, πάθος, "suffering", "passion" and , "producer of") in the oldest and broadest sense, is any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a germ ...
s, one of which is
humoral immunity. This defence mechanism produces
antibodies (large
glycoproteins
Glycoproteins are proteins which contain oligosaccharide chains covalently attached to amino acid side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosy ...
) in response to an immune stimulus. Many cells of the immune system are required for this process, including
lymphocyte
A lymphocyte is a type of white blood cell (leukocyte) in the immune system of most vertebrates. Lymphocytes include natural killer cells (which function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic ad ...
s (
T-cell
A T cell is a type of lymphocyte. T cells are one of the important white blood cells of the immune system and play a central role in the adaptive immune response. T cells can be distinguished from other lymphocytes by the presence of a T-cell r ...
s and
B-cells) and
antigen presenting cells. These cells coordinate an immune response upon the detection of foreign proteins (
antigen
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune respons ...
s), producing antibodies that bind to these antigens. In normal physiology, lymphocytes that recognise human proteins (
autoantigen
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". ...
s) either undergo programmed cell death (
apoptosis) or become non-functional. This
self-tolerance means that lymphocytes should not incite an immune response against human cellular antigens. Sometimes, however, this process malfunctions and antibodies are produced against human antigens, which may lead to autoimmune disease.
ANA subtypes
ANAs are found in many disorders, as well as some healthy individuals. These disorders include:
systemic lupus erythematosus (SLE),
rheumatoid arthritis
Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects joints. It typically results in warm, swollen, and painful joints. Pain and stiffness often worsen following rest. Most commonly, the wrist and hands are invol ...
,
Sjögren syndrome,
scleroderma
Scleroderma is a group of autoimmune diseases that may result in changes to the skin, blood vessels, muscles, and internal organs. The disease can be either localized to the skin or involve other organs, as well. Symptoms may include areas ...
,
polymyositis
Polymyositis (PM) is a type of chronic inflammation of the muscles (inflammatory myopathy) related to dermatomyositis and inclusion body myositis. Its name means "inflammation of many muscles" ('' poly-'' + '' myos-'' + '' -itis''). The inflam ...
,
dermatomyositis
Dermatomyositis (DM) is a long-term inflammatory disorder which affects skin and the muscles. Its symptoms are generally a skin rash and worsening muscle weakness over time. These may occur suddenly or develop over months. Other symptoms may inc ...
,
primary biliary cirrhosis,
drug induced lupus,
autoimmune hepatitis
Autoimmune hepatitis, formerly known as lupoid hepatitis, plasma cell hepatitis, or autoimmune chronic active hepatitis, is a chronic, autoimmune disease of the liver that occurs when the body's immune system attacks liver cells, causing the live ...
,
multiple sclerosis,
discoid lupus,
thyroid disease
Thyroid disease is a medical condition that affects the function of the thyroid gland. The thyroid gland is located at the front of the neck and produces thyroid hormones that travel through the blood to help regulate many other organs, meaning ...
,
antiphospholipid syndrome
Antiphospholipid syndrome, or antiphospholipid antibody syndrome (APS or APLS), is an autoimmune, hypercoagulable state caused by antiphospholipid antibodies. APS provokes blood clots (thrombosis) in both arteries and veins as well as pregnancy-r ...
,
juvenile idiopathic arthritis
Juvenile idiopathic arthritis (JIA) is the most common, chronic rheumatic disease of childhood, affecting approximately one per 1,000 children. ''Juvenile'', in this context, refers to disease onset before 16 years of age, while ''idiopathic'' r ...
,
psoriatic arthritis
Psoriatic arthritis is a long-term inflammatory arthritis that occurs in people affected by the autoimmune disease psoriasis. The classic feature of psoriatic arthritis is swelling of entire fingers and toes with a sausage-like appearance. Th ...
,
juvenile dermatomyositis
Juvenile dermatomyositis (JDM) is an idiopathic inflammatory myopathy (IMM) of presumed autoimmune dysfunction resulting in muscle weakness among other complications. It manifests itself in children; it is the pediatric counterpart of dermatomyo ...
,
idiopathic thrombocytopaenic purpura,
infection
An infection is the invasion of tissues by pathogens, their multiplication, and the reaction of host tissues to the infectious agent and the toxins they produce. An infectious disease, also known as a transmissible disease or communicable dis ...
and
cancer
Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
. These antibodies can be subdivided according to their specificity, and each subset has different propensities for specific disorders.
Extractable nuclear antigens
Extractable nuclear antigen 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, ...
s (ENA) are a group of
autoantigen
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". ...
s that were originally identified as antibody targets in people with autoimmune disorders. They are termed ENA because they can be extracted from the cell nucleus with saline.
The ENAs consist of
ribonucleoprotein
Nucleoproteins are proteins conjugated with nucleic acids (either DNA or RNA). Typical nucleoproteins include ribosomes, nucleosomes and viral nucleocapsid proteins.
Structures
Nucleoproteins tend to be positively charged, facilitating int ...
s and non-
histone
In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn a ...
proteins, named by either the name of the donor who provided the prototype serum (Sm, Ro, La, Jo), or the name of the disease setting in which the antibodies were found (SS-A, SS-B, Scl-70).
Anti-Ro/SS-A and anti-La/SS-B
Anti-Ro and
anti-La antibodies, also known as SS-A and SS-B, respectively, are commonly found in primary
Sjögren's syndrome, an
autoimmune disorder
An autoimmune disease is a condition arising from an abnormal immune response to a functioning body part. At least 80 types of autoimmune diseases have been identified, with some evidence suggesting that there may be more than 100 types. Nearly ...
that affects the
exocrine gland
Exocrine glands are glands that secrete substances on to an epithelial surface by way of a duct. Examples of exocrine glands include sweat, salivary, mammary, ceruminous, lacrimal, sebaceous, prostate and mucous. Exocrine glands are one of ...
s. The presence of both antibodies is found in 30–60% of Sjögren's syndrome, anti-Ro antibodies alone are found in 50–70% of Sjögren's syndrome and 30% of SLE with cutaneous involvement, and anti-La antibodies are rarely found in isolation.
Anti-La antibodies are also found in SLE; however, Sjögren's syndrome is normally also present.
Anti-Ro antibodies are also found less frequently in other disorders including autoimmune liver diseases,
coeliac disease
Coeliac disease (British English) or celiac disease (American English) is a long-term autoimmune disorder, primarily affecting the small intestine, where individuals develop intolerance to gluten, present in foods such as wheat, rye and barle ...
, autoimmune rheumatic diseases, cardiac neonatal
lupus erythematosus
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, blo ...
and
polymyositis
Polymyositis (PM) is a type of chronic inflammation of the muscles (inflammatory myopathy) related to dermatomyositis and inclusion body myositis. Its name means "inflammation of many muscles" ('' poly-'' + '' myos-'' + '' -itis''). The inflam ...
.
During pregnancy, anti-Ro antibodies can cross the
placenta
The placenta is a temporary embryonic and later fetal organ that begins developing from the blastocyst shortly after implantation. It plays critical roles in facilitating nutrient, gas and waste exchange between the physically separate mate ...
and cause
heart block
Heart block (HB) is a disorder in the heart's rhythm due to a fault in the natural pacemaker. This is caused by an obstruction – a block – in the electrical conduction system of the heart. Sometimes a disorder can be inherited. Despite the se ...
and neonatal lupus in babies.
In Sjögren's syndrome, anti-Ro and anti-La antibodies correlate with early onset, increased disease duration,
parotid gland
The parotid gland is a major salivary gland in many animals. In humans, the two parotid glands are present on either side of the mouth and in front of both ears. They are the largest of the salivary glands. Each parotid is wrapped around the ma ...
enlargement, disease outside the glands and infiltration of glands by lymphocytes.
Anti-Ro antibodies are specific to components of the Ro-RNP complex, comprising 45kDa, 52kDa, 54kDa and 60kDa proteins and
RNA. The 60kDa
DNA/RNA binding protein and 52kDa
T-cell
A T cell is a type of lymphocyte. T cells are one of the important white blood cells of the immune system and play a central role in the adaptive immune response. T cells can be distinguished from other lymphocytes by the presence of a T-cell r ...
regulatory protein are the best characterised antigens of anti-Ro antibodies. Collectively, these proteins are part of a
ribonucleoprotein
Nucleoproteins are proteins conjugated with nucleic acids (either DNA or RNA). Typical nucleoproteins include ribosomes, nucleosomes and viral nucleocapsid proteins.
Structures
Nucleoproteins tend to be positively charged, facilitating int ...
(RNP) complex that associate with the human
Y RNA
Y RNAs are small non-coding RNAs. They are components of the Ro60 ribonucleoprotein particle which is a target of autoimmune antibodies in patients with systemic lupus erythematosus. They are also reported to be necessary for DNA replication thr ...
s, hY1-hY5. The La antigen is a 48kDa
transcription
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
termination factor of
RNA polymerase III
In eukaryote cells, RNA polymerase III (also called Pol III) is a protein that transcribes DNA to synthesize ribosomal 5S rRNA, tRNA and other small RNAs.
The genes transcribed by RNA Pol III fall in the category of "housekeeping" genes whose e ...
, which associates with the Ro-RNP complex.
The mechanism of antibody production in Sjögren's syndrome is not fully understood, but
apoptosis (programmed cell death) and
molecular mimicry Molecular mimicry is defined as the theoretical possibility that sequence similarities between foreign and self-peptides are sufficient to result in the cross-activation of autoreactive T or B cells by pathogen-derived peptides. Despite the preva ...
may play a role.
The Ro and La antigens are expressed on the surface of cells undergoing apoptosis and may cause the inflammation within the salivary gland by interaction with cells of the immune system. The antibodies may also be produced through molecular mimicry, where cross reactive antibodies bind to both virus and human proteins. This may occur with one of the antigens, Ro or La, and may subsequently produce antibodies to other proteins through a process known as
epitope spreading
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". ...
. The retroviral gag protein shows similarity to the La protein and is proposed as a possible example for molecular mimicry in Sjögren's syndrome.
Anti-Sm
Anti-Smith (Anti-Sm) antibodies are a very specific marker for SLE. Approximately 99% of individuals without SLE lack anti-Sm antibodies, but only 20% of people with SLE have the antibodies. They are associated with
central nervous system
The central nervous system (CNS) is the part of the nervous system consisting primarily of the brain and spinal cord. The CNS is so named because the brain integrates the received information and coordinates and influences the activity of all p ...
involvement,
kidney disease,
lung fibrosis
Pulmonary fibrosis is a condition in which the lungs become scarred over time. Symptoms include shortness of breath, a dry cough, feeling tired, weight loss, and nail clubbing. Complications may include pulmonary hypertension, respiratory failu ...
and
pericarditis in SLE, but they are not associated with disease activity. The antigens of the anti-Sm antibodies are the core units of the
small nuclear ribonucleoprotein
snRNPs (pronounced "snurps"), or small nuclear ribonucleoproteins, are RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which RNA splicing, spli ...
s (snRNPs), termed A to G, and will bind to the U1, U2, U4, U5 and U6 snRNPs. Most commonly, the antibodies are specific for the B, B' and D units.
Molecular and epidemiological studies suggest that anti-Sm antibodies may be induced by molecular mimicry because the protein shows some similarity to
Epstein-Barr virus proteins.
Anti-nRNP/anti-U1-RNP
Anti-nuclear ribonucleoprotein (anti-nRNP) antibodies, also known as anti-U1-RNP antibodies, are found in 30–40% of SLE. They are often found with anti-Sm antibodies, but they may be associated with different clinical associations. In addition to SLE, these antibodies are highly associated with
mixed connective tissue disease
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 ...
. Anti-nRNP antibodies recognise the A and C core units of the snRNPs and because of this they primarily bind to the U1-snRNP.
The immune response to RNP may be caused by the presentation of the nuclear components on the cell membrane in apoptotic blebs. Molecular mimicry has also been suggested as a possible mechanism for the production of antibodies to these proteins because of similarity between U1-RNP polypeptides and Epstein-Barr virus polypeptides.
Anti-Scl-70/anti-topoisomerase I
Anti-Scl-70 antibodies
Anti-Scl-70 (also called anti-topoisomerase I after the type I topoisomerase target) is an anti-topoisomerase antibody-type of anti-nuclear autoantibodies, seen mainly in diffuse systemic scleroderma (with a sensitivity of 28–70%), but is als ...
are linked to
scleroderma
Scleroderma is a group of autoimmune diseases that may result in changes to the skin, blood vessels, muscles, and internal organs. The disease can be either localized to the skin or involve other organs, as well. Symptoms may include areas ...
.
The sensitivity of the antibodies for scleroderma is approximately 34%, but is higher for cases with diffuse cutaneous involvement (40%), and lower for limited cutaneous involvement (10%). The specificity of the antibodies is 98% and 99.6% in other rheumatic diseases and normal individuals, respectively.
In addition to scleroderma, these antibodies are found in approximately 5% of individuals with SLE.
The antigenic target of anti-Scl-70 antibodies is
topoisomerase I
DNA topoisomerases (or topoisomerases) are enzymes that catalyze changes in the topological state of DNA, interconverting relaxed and supercoiled forms, linked (catenated) and unlinked species, and knotted and unknotted DNA. Topological issues i ...
.
Anti-Jo-1
Although anti-Jo-1 antibodies are often included with ANAs, they are actually antibodies to the cytoplasmic protein,
Histidyl-tRNA synthetase - an aminoacyl-tRNA synthetase essential for the synthesis of histidine loaded tRNA.
They are highly associated with
polymyositis
Polymyositis (PM) is a type of chronic inflammation of the muscles (inflammatory myopathy) related to dermatomyositis and inclusion body myositis. Its name means "inflammation of many muscles" ('' poly-'' + '' myos-'' + '' -itis''). The inflam ...
and
dermatomyositis
Dermatomyositis (DM) is a long-term inflammatory disorder which affects skin and the muscles. Its symptoms are generally a skin rash and worsening muscle weakness over time. These may occur suddenly or develop over months. Other symptoms may inc ...
, and are rarely found in other connective tissue diseases. Around 20–40% of polymyositis is positive for Jo-1 antibodies and most will have interstitial lung disease, HLA-DR3 and HLA-DRw52
human leukocyte antigen
The human leukocyte antigen (HLA) system or complex is a complex of genes on chromosome 6 in humans which encode cell-surface proteins responsible for the regulation of the immune system. The HLA system is also known as the human version of th ...
(HLA) markers; collectively known as Jo-1 syndrome.
Anti-dsDNA
Anti-double stranded DNA (anti-dsDNA) antibodies are highly associated with SLE. They are a very
specific
Specific may refer to:
* Specificity (disambiguation)
* Specific, a cure or therapy for a specific illness
Law
* Specific deterrence, focussed on an individual
* Specific finding, intermediate verdict used by a jury in determining the fina ...
marker for the disease, with some studies quoting nearly 100%.
Data on
sensitivity ranges from 25 to 85%. Anti-dsDNA antibody levels, known as titres, correlate with disease activity in SLE; high levels indicate more active lupus. The presence of anti-dsDNA antibodies is also linked with
lupus nephritis
Lupus nephritis is an inflammation of the kidneys caused by systemic lupus erythematosus (SLE), an autoimmune disease. It is a type of glomerulonephritis in which the glomeruli become inflamed. Since it is a result of SLE, this type of glomerulo ...
and there is evidence they are the cause. Some anti-dsDNA antibodies are cross reactive with other antigens found on the
glomerular basement membrane (GBM) of the kidney, such as
heparan sulphate
Heparan sulfate (HS) is a linear polysaccharide found in all animal tissues. It occurs as a proteoglycan (HSPG, i.e. Heparan Sulfate ProteoGlycan) in which two or three HS chains are attached in close proximity to cell surface or extracellular ...
,
collagen IV,
fibronectin
Fibronectin is a high- molecular weight (~500-~600 kDa) glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteins called integrins. Fibronectin also binds to other extracellular matrix proteins such as collage ...
and
laminin
Laminins are a family of glycoproteins of the extracellular matrix of all animals. They are major components of the basal lamina (one of the layers of the basement membrane), the protein network foundation for most cells and organs. The laminins ...
. Binding to these antigens within the kidney could cause
inflammation
Inflammation (from la, inflammatio) is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, and is a protective response involving immune cells, blood vessels, and molec ...
and
complement fixation, resulting in kidney damage. Presence of high DNA-binding and low
C3 levels have been shown to have extremely high predictive value (94%) for the diagnosis of SLE.
It is also possible that the anti-dsDNA antibodies are internalised by cells when they bind membrane antigens and then are displayed on the cell surface. This could promote inflammatory responses by T-cells within the kidney. It is important to note that not all anti-dsDNA antibodies are associated with lupus nephritis and that other factors can cause this symptom in their absence. The antigen of anti-dsDNA antibodies is
double stranded DNA.
Anti-histone antibodies
Anti-histone antibodies 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 i ...
are found in the serum of up to 75–95% of people with
drug induced lupus and 75% of idiopathic SLE. Unlike anti-dsDNA antibodies in SLE, these antibodies do not fix complement. Although they are most commonly found in drug induced lupus, they are also found in some cases of SLE,
scleroderma
Scleroderma is a group of autoimmune diseases that may result in changes to the skin, blood vessels, muscles, and internal organs. The disease can be either localized to the skin or involve other organs, as well. Symptoms may include areas ...
,
rheumatoid arthritis
Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects joints. It typically results in warm, swollen, and painful joints. Pain and stiffness often worsen following rest. Most commonly, the wrist and hands are invol ...
and
undifferentiated connective tissue disease
Undifferentiated connective tissue disease (UCTD) is a disease in which the body mistakenly attacks its own tissues. It is diagnosed when there is evidence of an existing autoimmune condition which does not meet the criteria for any specific auto ...
. Many drugs are known to cause drug induced lupus and they produce various antigenic targets within the nucleosome that are often cross reactive with several histone proteins and DNA.
Procainamide
Procainamide (PCA) is a medication of the antiarrhythmic class used for the treatment of cardiac arrhythmias. It is classified by the Vaughan Williams classification system as class Ia; thus it is a sodium channel blocker of cardiomyocytes. In add ...
causes a form of drug-induced lupus that produces antibodies to the histone H2A and H2B complex.
Anti-gp210 and anti-p62
Both
anti-glycoprotein-210 (anti-gp210) and
anti-nucleoporin 62 (anti-p62) antibodies are antibodies to components of the nuclear membrane and are found in
primary biliary cirrhosis (PBC). Each antibody is present in approximately 25–30% of PBC. The antigens of both antibodies are constituents of the
nuclear membrane
The nuclear envelope, also known as the nuclear membrane, is made up of two lipid bilayer membranes that in eukaryotic cells surround the nucleus, which encloses the genetic material.
The nuclear envelope consists of two lipid bilayer membra ...
. gp210 is a 200kDa protein involved in anchoring components of the
nuclear pore
A nuclear pore is a part of a large complex of proteins, known as a nuclear pore complex that spans the nuclear envelope, which is the double membrane surrounding the eukaryotic cell nucleus. There are approximately 1,000 nuclear pore complexe ...
to the nuclear membrane. The p62 antigen is a 60kDa nuclear pore complex.
Anti-centromere antibodies
Anti-centromere antibodies
Anti-centromere antibodies (ACAs; often styled solid, anticentromere) are autoantibodies specific to centromere and kinetochore function. They occur in some autoimmune diseases, frequently in limited systemic scleroderma (formerly called CREST sy ...
are associated with limited cutaneous systemic sclerosis, also known as
CREST syndrome
CREST syndrome, also known as the limited cutaneous form of systemic sclerosis (lcSSc), is a multisystem connective tissue disorder. The acronym "CREST" refers to the five main features: calcinosis, Raynaud's phenomenon, esophageal dysmotility, ...
, primary biliary cirrhosis and proximal scleroderma.
There are six known antigens, which are all associated with the
centromere
The centromere links a pair of sister chromatids together during cell division. This constricted region of chromosome connects the sister chromatids, creating a short arm (p) and a long arm (q) on the chromatids. During mitosis, spindle fibers ...
; CENP-A to CENP-F. CENP-A is a 17kDa
histone H3
Histone H3 is one of the five main histones involved in the structure of chromatin in eukaryotic cells. Featuring a main globular domain and a long N-terminal tail, H3 is involved with the structure of the nucleosomes of the 'beads on a stri ...
-like protein. CENP-B is an 80kDa DNA binding protein involved in the folding of
heterochromatin. CENP-C is a 140kDa protein involved in
kinetochore
A kinetochore (, ) is a disc-shaped protein structure associated with duplicated chromatids in eukaryotic cells where the spindle fibers attach during cell division to pull sister chromatids apart. The kinetochore assembles on the centromere and ...
assembly. CENP-D is a 50kDa protein of unknown function, but may be
homologous to another protein involved in
chromatin condensation
Prophase () is the first stage of cell division in both mitosis and meiosis. Beginning after interphase, DNA has already been replicated when the cell enters prophase. The main occurrences in prophase are the condensation of the chromatin reti ...
,
RCC1
Regulator of chromosome condensation 1, also known as RCC1, Ran guanine nucleotide exchange factor and RanGEF, is the name for a human gene and protein.
RCC1 also functions as a guanine nucleotide exchange factor for Ran GTPase.
Interactions
...
. CENP-E is a 312kDa protein from the
kinesin
A kinesin is a protein belonging to a class of motor proteins found in eukaryotic cells.
Kinesins move along microtubule (MT) filaments and are powered by the hydrolysis of adenosine triphosphate (ATP) (thus kinesins are ATPases, a type of enzy ...
motor protein family. CENP-F is a 367kDa protein from the nuclear matrix that associates with the kinetochore in late
G2 phase
G2 phase, Gap 2 phase, or Growth 2 phase, is the third subphase of interphase in the cell cycle directly preceding mitosis. It follows the successful completion of S phase, during which the cell’s DNA is replicated. G2 phase ends with the ...
during mitosis. CENP-A, B and C antibodies are most commonly found (16–42% of systemic sclerosis) and are associated with Raynaud's phenomenon,
telangiectasia
Telangiectasias, also known as spider veins, are small dilated blood vessels that can occur near the surface of the skin or mucous membranes, measuring between 0.5 and 1 millimeter in diameter. These dilated blood vessels can develop anywhere on ...
s, lung involvement and early onset in systemic sclerosis.
Anti-sp100
Anti-sp100 antibodies are found in approximately 20–30% of
primary biliary cirrhosis (PBC). They are found in few individuals without PBC, and therefore are a very specific marker of the disease. The sp100 antigen is found within nuclear bodies; large protein complexes in the nucleus that may have a role in cell growth and differentiation.
Anti-PM-Scl
Anti-PM-Scl antibodies are found in up to 50% of
polymyositis/systemic sclerosis (PM/SSc) overlap syndrome. Around 80% of individuals with antibodies present in their blood serum will have the disorder. The presence of the antibodies is linked to limited cutaneous involvement of PM/SSc overlap syndrome. The antigenic targets of the antibodies are components of the
RNA-processing
exosome complex
The exosome complex (or PM/Scl complex, often just called the exosome) is a multi-protein intracellular Protein complex, complex capable of degrading various types of RNA (ribonucleic acid) molecules. Exosome complexes are found in both eukaryo ...
in the
nucleolus
The nucleolus (, plural: nucleoli ) is the largest structure in the nucleus of eukaryotic cells. It is best known as the site of ribosome biogenesis, which is the synthesis of ribosomes. The nucleolus also participates in the formation of ...
.
There are ten proteins in this complex and antibodies to eight of them are found at varying frequencies; PM/Scl-100 (70–80%), PM/Scl-75 (46–80%), hRrp4 (50%), hRrp42 (21%), hRrp46 (18%), hCs14 (14%), hRrp41 (10%) and hRrp40 (7%).
Anti-DFS70 antibodies
Anti-DFS70 antibodies generate a dense fine speckled pattern in indirect immunofluorescence and are found in normals and in various conditions, but are not associated with a systemic autoimmune pathology. Therefore, they can be used to help to rule out such conditions in ANA positive individuals. A significant number of patients are diagnosed as systemic lupus erythematosus or undifferentiated connective tissue disease largely based on a positive ANA. In case no defined autoantibody can be detected (e.g. anti-ENA antibodies), the testing of anti-DFS70 antibodies is recommended to verify the diagnosis. Anti-DFS70 antibody tests are available as CE-marked tests. Until now, no FDA cleared assay is available.
ANA test
![ANA Immunofluorescence](https://upload.wikimedia.org/wikipedia/commons/d/d6/ANA_Immunofluorescence.png)
The presence of ANAs in blood can be confirmed by a screening test. Although there are many tests for the detection of ANAs, the most common tests used for screening are indirect immunofluorescence and
enzyme-linked immunosorbent assay
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 ...
(ELISA).
Following detection of ANAs, various subtypes are determined.
Indirect immunofluorescence
Indirect immunofluorescence is one of the most commonly used tests for ANAs. Typically, HEp-2 cells are used as a substrate to detect the antibodies in human serum. Microscope slides are coated with HEp-2 cells and the serum is incubated with the cells. If the said and targeted antibodies are present then they will bind to the
antigen
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune respons ...
s on the cells; in the case of ANAs, the antibodies will bind to the nucleus. These can be visualised by adding a fluorescent tagged (usually
FITC or rhodopsin B) anti-human antibody that binds to the antibodies. The molecule will fluoresce when a specific wavelength of light shines on it, which can be seen under the microscope. Depending on the antibody present in the human serum and the localisation of the antigen in the cell, distinct patterns of fluorescence will be seen on the HEp-2 cells.
Levels of antibodies are analysed by performing dilutions on blood serum. An ANA test is considered positive if fluorescence is seen at a titre of 1:40/1:80. Higher titres are more clinically significant as low positives (≤1:160) are found in up to 20% of healthy individuals, especially the elderly. Only around 5% of the healthy population have ANA titres of 1:160 or higher.
HEp-2
![ANA NUCLEOLAR 3](https://upload.wikimedia.org/wikipedia/commons/5/53/ANA_NUCLEOLAR_3.jpg)
Until around 1975, when HEp-2 cells were introduced, animal tissue was used as the standard substrate for immunofluorescence.
HEp-2 cells are currently one of the most common substrates for ANA detection by immunofluorescence.
Originally started a laryngeal carcinoma strain, the cell line was contaminated and displaced by
HeLa
HeLa (; also Hela or hela) is an immortalized cell line used in scientific research. It is the oldest and most commonly used human cell line. The line is derived from cervical cancer cells taken on February 8, 1951, named after Henrietta ...
cells, and has now been identified as actually HeLa cells.
They are superior to the previously used animal tissues because of their large size and the high rate of
mitosis (cell division) in the
cell line. This allows the detection of antibodies to mitosis-specific antigens, such as centromere antibodies. They also allow identification of anti-Ro antibodies, because
acetone
Acetone (2-propanone or dimethyl ketone), is an organic compound with the formula . It is the simplest and smallest ketone (). It is a colorless, highly volatile and flammable liquid with a characteristic pungent odour.
Acetone is miscib ...
is used for
fixation of the cells (other fixatives can wash the antigen away).
There are many nuclear staining patterns seen on HEp-2 cells: homogeneous, speckled, nucleolar, nuclear membranous, centromeric, nuclear dot and pleomorphic. The homogeneous pattern is seen when the condensed
chromosomes
A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
and interphase
chromatin
Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in r ...
stain. This pattern is associated with
anti-dsDNA 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 p ...
, antibodies to nucleosomal components, and anti-histone antibodies. There are two speckled patterns: fine and coarse. The fine speckled pattern has fine nuclear staining with unstained
metaphase
Metaphase ( and ) is a stage of mitosis in the eukaryotic cell cycle in which chromosomes are at their second-most condensed and coiled stage (they are at their most condensed in anaphase). These chromosomes, carrying genetic information, alig ...
chromatin, which is associated with anti-Ro and anti-La antibodies. The coarse staining pattern has coarse granular nuclear staining, caused by anti-U1-RNP and anti-Sm antibodies. The nucleolar staining pattern is associated with many antibodies including anti-Scl-70, anti-PM-Scl, anti-fibrillarin and anti-Th/To. Nuclear membrane staining appears as a fluorescent ring around the cell nucleus and are produced by anti-gp210 and anti-p62 antibodies. The centromere pattern shows multiple
nuclear dots in interphase and mitotic cells, corresponding to the number of chromosomes in the cell. Nuclear dot patterns show between 13 and 25 nuclear dots in
interphase
Interphase is the portion of the cell cycle that is not accompanied by visible changes under the microscope, and includes the G1, S and G2 phases. During interphase, the cell grows (G1), replicates its DNA (S) and prepares for mitosis (G2). A c ...
cells and are produced by anti-
sp100 antibodies. Pleomorphic pattern is caused by antibodies to the
proliferating cell nuclear antigen
Proliferating cell nuclear antigen (PCNA) is a DNA clamp that acts as a processivity factor for DNA polymerase δ in eukaryotic cells and is essential for replication. PCNA is a homotrimer and achieves its processivity by encircling the DNA, wh ...
.
Indirect immunofluorescence has been shown to be slightly superior compared to ELISA in detection of ANA from HEp-2 cells.
''Crithidia luciliae''
![CRITHIDIA 2](https://upload.wikimedia.org/wikipedia/commons/3/36/CRITHIDIA_2.jpg)
''
Crithidia luciliae'' are
haemoflaggelate single celled protist
A protist () is any eukaryotic organism (that is, an organism whose cells contain a cell nucleus) that is not an animal, plant, or fungus. While it is likely that protists share a common ancestor (the last eukaryotic common ancestor), the exc ...
s. They are used as a substrate in immunofluorescence for the detection of anti-dsDNA antibodies. They possess an
organelle known as the
kinetoplast
A kinetoplast is a network of circular DNA (called kDNA) inside a large mitochondrion that contains many copies of the mitochondrial genome. The most common kinetoplast structure is a disk, but they have been observed in other arrangements. Kinet ...
which is a large
mitochondrion with a network of interlocking circular dsDNA molecules. After incubation with serum containing anti-dsDNA antibodies and fluorescent-labelled anti-human antibodies, the kinetoplast will fluoresce. The lack of other nuclear antigens in this organelle means that using ''C. luciliae'' as a substrate allows for the specific detection of anti-dsDNA antibodies.
ELISA
Enzyme-linked immunosorbent assay
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 ...
(ELISA) uses antigen-coated
microtitre plate
A microplate, also known as a microtiter plate (''Microtiter'' is a registered trademark in the United States, therefore it should not be used generically without attribution), microwell plate or multiwell, is a flat plate with multiple "wells" ...
s for the detection of ANAs.
Each well of a microtitre plate is coated with either a single antigen or multiple antigens to detect specific antibodies or to screen for ANAs, respectively. The antigens are either from cell extracts or recombinant. Blood serum is incubated in the wells of the plate and is washed out. If antibodies that bind to antigen are present then they will remain after washing. A secondary anti-human antibody conjugated to an enzyme such as
horseradish peroxidase is added. The enzyme reaction will produce a change in colour of the solution that is proportional to the amount of antibody bound to the antigen.
There are significant differences in the detection of ANA by immunofluorescence and different ELISA kits and there is only a marginal agreement between these. A clinician must be familiar with the differences in order to evaluate the outcomes of the various assays.
Sensitivity
The following table lists the
sensitivity of different types of ANAs for different diseases.
Some ANAs appear in several types of disease, resulting in lower
specificity of the test. For example, IgM-
rheumatoid factor (IgM-RF) have been shown to cross-react with ANA giving falsely positive
immunofluorescence.
Positive ANA as well as anti-DNA antibodies have been reported in patients with
autoimmune thyroid disease.
ANA can have a positive test result in up to 45% of people with autoimmune thyroid conditions or
rheumatoid arthritis
Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects joints. It typically results in warm, swollen, and painful joints. Pain and stiffness often worsen following rest. Most commonly, the wrist and hands are invol ...
and up to 15% of people with
HIV
The human immunodeficiency viruses (HIV) are two species of ''Lentivirus'' (a subgroup of retrovirus) that infect humans. Over time, they cause acquired immunodeficiency syndrome (AIDS), a condition in which progressive failure of the immune ...
or
hepatitis C.
As per
Lupus Foundation of America
The Lupus Foundation of America (LFA), founded in 1977, is a national voluntary health organization based in Washington, D.C., with a network of chapters, offices and support groups located in communities throughout the United States. The Found ...
, "about 5% of the general population will have a positive ANA. However, at least 95% of the people who have a positive ANA do not have lupus. A positive ANA test can sometimes run in families, even if family members have no evidence of lupus."
On the other hand, they say, although 95% of the patients who actually have lupus test positive for ANA, "Only a small percentage have a negative ANA, and many of those have other antibodies (such as
anti-phospholipid antibodies
Antiphospholipid syndrome, or antiphospholipid antibody syndrome (APS or APLS), is an autoimmune, hypercoagulable state caused by antiphospholipid antibodies. APS provokes blood clots (thrombosis) in both arteries and veins as well as pregnancy-r ...
, anti-Ro, anti-SSA) or their ANA converted from positive to negative from
steroids,
cytotoxic medications, or
uremia
Uremia is the term for high levels of urea in the blood. Urea is one of the primary components of urine. It can be defined as an excess of amino acid and protein metabolism end products, such as urea and creatinine, in the blood that would be no ...
(kidney failure)."
History
![LECell](https://upload.wikimedia.org/wikipedia/commons/e/eb/LECell.jpg)
The
LE cell was discovered in
bone marrow in 1948 by Hargraves ''et al.''
[Hargraves M, Richmond H, Morton R. ''Presentation of two bone marrow components, the tart cell and the LE cell.'' Mayo Clin Proc 1948;27:25–28.] In 1957 Holborow et al. first demonstrated ANA using indirect immunofluorescence.
This was the first indication that processes affecting the cell nucleus were responsible for SLE. In 1959 it was discovered that serum from individuals with SLE contained antibodies that precipitated with saline extracts of nuclei, known as
extractable nuclear antigens 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, ...
(ENAs). This led to the characterisation of ENA antigens and their respective antibodies. Thus, anti-Sm and anti-RNP antibodies were discovered in 1966 and 1971, respectively. In the 1970s, the anti-Ro/anti-SS-A and anti-La/anti-SS-B antibodies were discovered. The Scl-70 antibody was known to be a specific antibody to scleroderma in 1979, however the antigen (topoisomerase-I) was not characterised until 1986. The Jo-1 antigen and antibody were characterised in 1980.
See also
*
Anti-neutrophil cytoplasmic antibody
Anti-neutrophil cytoplasmic antibodies (ANCAs) are a group of autoantibodies, mainly of the IgG type, against antigens in the cytoplasm of neutrophils (the most common type of white blood cell) and monocytes. They are detected as a blood t ...
(ANCA)
*
Rheumatoid factor
References
External links
Autoimmunityblog – HEp-2 ANA summary
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{{Autoantibodies
Chemical pathology
Autoantibodies
Antibodies
Immunologic tests