ANA ELISA

Antibodies capable of binding to certain structures

within the nucleus

Found when immune system maybe predisposed to

cause inflammation against self-tissues

IgG, IgM and IgA

Possibility of autoimmune disease

 Determines the presence of antibodies which react to

various parts of the nucleus (Anti-nuclear)

Test

Previously called Fluorescent Antinuclear Antibody (FANA) 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 attach

to complexes formed

Cells bounded with ANA fluoresces

***This test is neither specific nor sensitive

 Homogenous / Diffuse

nucleoprotein; Systemic Lupus Erythematosus and Mixed Connective Tissue Disease

Peripheral or Rim DNA;

Scleroderma

 Speckled Nuclear antigens;

Systemic Lupus Erythematosus, Sjogrens Syndrome and Rheumatoid Arthritis

Nucleolar Nucleolus;

Scleroderma and Polymyositis

DISEASES

 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.

 Signs and Symptoms

Joint and muscle pains

small joints of the hands and feet tend to be the ones affected

most 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 fingers and toes) Alopecia (hair loss) Blood and lymph mild anemia

 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 can

lead to the kidneys leaking protein and blood into the urine, suggestive of poor kidney function renal failure uncommon Brain and nervous system mild depression and anxiety

 autoimmune disease

involves inflammation of glands and other tissues in

the 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.

 Scleroderma is a disease associated with hardening of

the skin caused by overproduction of collagen.

Signs and Symptoms
skin thickening spontaneous scarring blood vessel disease varying degrees of inflammation, associated with an

overactive immune system.

 It is a mixture of diseases of the connective tissue:

systemic lupus erythematosus, scleroderma, and polymyositis

Signs and Symptoms
very high blood levels of antinuclear antibodies (ANAs)

and antibodies to ribonucleoprotein (anti-RNP).

 Rheumatoid arthritis is a disease associated with the

inflammation 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

 Myositis is a disease associated with Inflammation and

degeneration 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

TEST

 Prepare HRP washed buffer, 2ml concentration and 78

ml 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 positive

negative control and patient sample

 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

 Incubate in the dark for 30 mins at room temperature 100 uL of HRP stop solution Read optical path within an hour at 450nm

 PRINCIPLE

Analyte Detected Anti-Nuclear Antibodies

(ANA)
Anti-chromatin Anti-dsDNA Targets dsDNA Specific marker for SLE 40-60% Reacts with antigens of the glomerular basement membrane Kidney damage

Anti-histones
Targets histones

 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%

 Anti-La RNPs(La antigen)

*both Anti-Ro and Anti-La- 30-60% of Px with Sjogrens Syndrome

*correlates with early onset, increased disease duration, parotid gland enlargement, infiltration by lymphocytes

 Anti-Scl-70 Type I Topoisomerase Associated with scleroderma - *10-40% Anti-centromere Centromeric proteins

Associated with CREST syndrome, primary billiary

cirrhosis, proximal scleroderma Anti-PCNA

 Anti-Jo-1 Histidine-tRNA ligase Associated with polymyositis (20-40%) and

dermatomyositis Anti-M2 Associated with primary billiary cirrhosis Anti-ribosomal P

 ANTIGENS bound to the surface of the microtiter

plate

Chromatin dsDNA histones Sm/RNP (Smith/rubonucleoprotein) SS-A SS-B Scl-70 Centromere PCNA Jo-1 M2 ribosomal-p protein

 average absorbance for each duplicates were first

determined
Sample Value (units) = (Absorbance/Low Positive) X Low Positive

Result Negative Positive Strong Positive

Units <20 20-60 >60

Microtiter Plate Negative Control Low Positive/ High Positive

Sample Diluent
HRP Wash Buffer HS HRP IgG conjugate TMB Chromogen

Polysterene with nuclear and cytoplasmic antigens Human serum (-) for Ab to nuclear and cytoplasmic antigens + Preservative Human serum (+) for Ab to nuclear and cytoplasmic antigens Tris-buffered saline, Tween 20, protein stabilizers and preservatives Tris-buffered saline, Tween 20 Anti-human IgG (goat), protein stabilizers and preservatives 3,3,5,5- Tetramethylbenzidine

HRP Stop Solution

0.344M Sulfuric Acid

OTHER TEST

 one of the most commonly used tests for ANAs. HEp-2 cells - are used as a substrate to detect the

antibodies in human serum.

Microscope slides - coated with HEp-2 cells and the

serum is incubated with the cells.

If antibodies are present then they will bind to

the antigens 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. Positive: fluorescence is seen at a titer of 1:40/1:80. Higher titers are more clinically significant as low positives (1:160)
HEp-2 cells are superior to the previously used animal tissues because of their large size and the high rate of mitosis in the cell line.