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ANA ELISA
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Antibodies capable of binding to certain structureswithin the nucleus
Found when immune system maybe predisposed to
cause inflammation against self-tissues
IgG, IgM and IgA
Possibility of autoimmune disease
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Determines the presence of antibodies which react tovarious parts of the nucleus (Anti-nuclear)
Previously called Fluorescent Antinuclear Antibody (FANA)Test
Serum is overlayed in a commercially prepared cell Rat liver Mouse kidney Cultured cells: Hep-2 cells and Hela cells
Second Antibody, tagged with a fluorescent dye, will attachto complexes formed
Cells bounded with ANA fluoresces
***This test is neither specific nor sensitive
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Homogenous / Diffuse nucleoprotein; Systemic LupusErythematosus and MixedConnective Tissue Disease
Peripheral or Rim DNA;Scleroderma
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Speckled Nuclear antigens;Systemic LupusErythematosus, SjogrensSyndrome and Rheumatoid
Arthritis
Nucleolar Nucleolus;Scleroderma and Polymyositis
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DISEASES
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autoimmune disease
characterized by acute and chronic inflammation of
various tissues of the body.
cause of SLE is unknown, however, heredity, viruses,
ultraviolet light, and drugs all may play some role.
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Signs and Symptoms Joint and muscle pains
small joints of the hands and feet tend to be the ones affectedmost
joint stiffness is common
Skin, mouth and hair Butterfly Rash (red rash which develops over the cheeks and
nose)
Raynaud's phenomenon (blood vessels just under the skin
may also be affected and cause poor circulation to the fingersand toes)
Alopecia (hair loss)
Blood and lymph
mild anemia
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Signs and Symptoms Heart and lungs
Pleurisy (pains in the side of the chest) Pericarditis (central chest pains)
Lung inflammation Kidneys
may develop inflammation of the kidneys, which canlead to the kidneys leaking protein and blood into theurine, suggestive of poor kidney function
renal failure uncommon Brain and nervous system
mild depression and anxiety
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autoimmune disease
involves inflammation of glands and other tissues inthe body.
Signs and Symptoms Dryness of eyes and mouth
Inflammation of lacrimal glands leads to water
production for tears whick leads to dry eyes Inflammation of salivary glands including the parotid
gland leads to dry mouth and lips.
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Scleroderma is a disease associated with hardening ofthe skin caused by overproduction of collagen.
Signs and Symptoms skin thickening
spontaneous scarring
blood vessel disease
varying degrees of inflammation, associated with anoveractive immune system.
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It is a mixture of diseases of the connective tissue:systemic lupus erythematosus, scleroderma, andpolymyositis
Signs and Symptoms very high blood levels of antinuclear antibodies (ANAs)
and antibodies to ribonucleoprotein (anti-RNP).
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Rheumatoid arthritis is a disease associated with theinflammation of the lining of the joints.
Signs and Symptoms Painful, swollen, tender, stiff joints (pain felt in the hands, wrists, elbows, feet ankles,
knees, or neck) Fatigue
loss of appetite Weight loss Mild fever
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Myositis is a disease associated with Inflammation anddegeneration of muscle tissues
Signs and Symptoms Weakness of the muscles
Inflammation of muscles
Difficulty in swallowing food
Abdominal cramps Presence of blood in feces
Weight loss, stiffness, fever, loss of voice
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TEST
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Prepare HRP washed buffer, 2ml concentration and 78ml distilled water
Sample dilution (1:41), 10 uL sample + 400 uL sample
diluents
Microtiter well with nuclear and cytoplasmic antigen
100 uL of prediluted low positive, high positivenegative control and patient sample
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Incubate for 30 mins at room temperature
Wash three times (200-300 uL of HRP washed buffer)
100 uL of HsHRP IgG conjugate
Repeat steps 3 and 4
100 uL TMB chromogen
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Incubate in the dark for 30 mins at room temperature
100 uL of HRP stop solution
Read optical path within an hour at 450nm
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PRINCIPLE
Analyte Detected Anti-Nuclear Antibodies(ANA)
Anti-chromatinAnti-dsDNA
Targets dsDNA
Specific marker for SLE 40-60%
Reacts with antigens of the glomerular basement membrane -
Kidney damageAnti-histones
Targets histones
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Anti-Sm/RNP Targets core protein of snRNPs
SLE *99% with anti-Sm/RNP (+) for SLE
--*20% of SLE px (+) for anti-Sm/RNP Not associated with disease activity
Associated with CNS involvement, kidney disease,lung fibrosis and pericarditis
Anti-Ro
Ro-RNP complex
Sjogrens Syndrome-50-70%
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Anti-La RNPs(La antigen)
*both Anti-Ro and Anti-La- 30-60% of Px with
Sjogrens Syndrome*correlates with early onset, increased diseaseduration, parotid gland enlargement, infiltrationby lymphocytes
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Anti-Scl-70 Type I Topoisomerase
Associated with scleroderma - *10-40%
Anti-centromere
Centromeric proteins
Associated with CREST syndrome, primary billiarycirrhosis, proximal scleroderma
Anti-PCNA
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Anti-Jo-1 Histidine-tRNA ligase
Associated with polymyositis (20-40%) anddermatomyositis
Anti-M2Associated with primary billiary cirrhosis
Anti-ribosomal P
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ANTIGENS bound to the surface of the microtiterplate Chromatin
dsDNA histones
Sm/RNP (Smith/rubonucleoprotein) SS-A SS-B Scl-70 Centromere
PCNA Jo-1 M2 ribosomal-p protein
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average absorbance for each duplicates were firstdetermined
Sample Value(units)
= (Absorbance/Low Positive) X Low Positive
Result UnitsNegative 60
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Microtiter Plate Polysterene with nuclear and cytoplasmic
antigens
Negative Control Human serum (-) for Ab to nuclear and
cytoplasmic antigens + Preservative
Low Positive/ High Positive Human serum (+) for Ab to nuclear and
cytoplasmic antigensSample Diluent Tris-buffered saline, Tween 20, protein
stabilizers and preservatives
HRP Wash Buffer Tris-buffered saline, Tween 20
HS HRP IgG conjugate Anti-human IgG (goat), protein stabilizers
and preservatives
TMB Chromogen 3,3,5,5- Tetramethylbenzidine
HRP Stop Solution 0.344M Sulfuric Acid
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OTHER TEST
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one of the most commonly used tests for ANAs.
HEp-2 cells - are used as a substrate to detect theantibodies in human serum.
Microscope slides - coated with HEp-2 cells and theserum is incubated with the cells.
If antibodies are present then they will bind tothe antigens on the cells; in the case of ANAs, theantibodies will bind to the nucleus.
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These can be visualised by adding a fluorescent tagged(usuallyFITC or Rhodopsin B) anti-human antibodythat binds to the antibodies.
Positive: fluorescence is seen at a titer of 1:40/1:80.
Higher titers are more clinically significant as lowpositives (1:160)
HEp-2 cells are superior to the previously usedanimal tissues because of their large size andthe high rate of mitosis in the cell line.
http://en.wikipedia.org/wiki/File:ANA_NUCLEOLAR_3.jpghttp://en.wikipedia.org/wiki/File:ANA_NUCLEOLAR_3.jpg
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