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7/28/2019 Quanta Lite

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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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