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1. Detection of specific Ab in patients with syphilis

2. The presence of auto – Ab that react to specific structures (Ag) in either
frozen section or culture of cells (more good to obtain nuclear cells
compared to frozen tissue section)

Indirect immunofluorescence assays for antinuclear antibodies (ANA) –

substrate, Ag includes:
1. Micro organism
2. Tissue culture line (HEP – 2)
3. Tissue culture line infected with particular type of virus
4. Frozen section of specific organ (rodent kidney or liver)

Antinuclear antibodies (ANA) test (indirect immunofluorescence)

1. Dilute the sample (serum)
2. Add to substrate (Ag)
3. Incubate in room temperature for 20 – 30 minutes in moisture chamber
4. Wash with tape water (gently) and followed by buffer 3X
5. Add antisera against human Ig (conjugate with FITC)
6. Incubate for 20 – 30 minutes at room temperature in moist chamber
7. Wash by water and followed by buffer 3X
8. Mount
9. Examine under fluorescence microscope

The most important Ag in nucleus is found in nucleoplasm:

1. Chromosomes with their DNA
2. Basic nuclear histone protein
3. Acid non histone unclear protein
4. Centomeric protein
5. mRNA
6. Nucleolus (RNA) is important Ag for patient with progressive systemic
sclerosis (scleroderma)
7. The nuclear membrane itself is antigenic

Four pattern of nuclear staining have been described:
1. Only peripheral or rim pattern (DNA pattern)
• Shows the most specific traditional ANA pattern for SLE
• Have two form that was differentiated by staining of chromosome
(chromosome pattern) or not
2. Homogenous pattern
• Specific for SLE
3. Speckled pattern (dot-dot)
• The least specific for SLE
• Also have two form that was differentiated by staining of
chromosome (chromosome pattern) or not
4. Nucleolar pattern (inside nucleus)
• Anti ribo-nucleoprotein (anti – RNP) in progressive systemic
sclerosis (scleroderma)

Anti neutrophil cytoplasmic Ab (ANCA) test:

1. Specificity for Wegener’s granulomatosis (WG) (WG – systemic
autoimmune disease)
2. Substrate (Ag): human neutrophil
3. Three staining pattern can be seen in neutrophil
• Granular cytoplasmic staining (C – ANCA) is highly specific for
• Peri – nuclear pattern (P – ANCA) is due to anti – myeloperoxidase
(less specific)
• ANA that may be seen in patient with SLE

Anti – DNA:
1. Ab against ds DNA were more specific for SLE
2. Immunofluorescence test used hemoflagellate (Crithidia luciliae) as
• Kinetoblast contains ds DNA with no ss DNA
• Nucleus: both ss DNA and ds DNA (highly specific for SLE)

Proliferating cell nuclear Ag (PCNA)

1. Anti – PCNA is specific for SLE
2. The Ag is found mainly in proliferating cells

Disease associated with anti – nuclear antibodies
1. MCTD: mix connective tissue disease
2. SLE: systemic lupus erythematosus
3. Sjorgen syndrome
4. Scleroderma

MCTD (Mixed Connective Tissue Disease):

1. Patient have only anti – ribonuclear protein (anti – RNP) auto Ab in high
2. Have high incidence of Ragnaud’s phenomenon (pain and colour changes
in fingers on exposure to cold)

PSS (Scleroderma):
1. Replacement of smooth muscle throughout the body by fibrous
connective tissue
2. Nucleolar ANA (ribonucleoprotein) pattern was associated with PSS

Sjorgen syndrome:
1. Dry eyes, dry mouth, and arthritis (inflammation of the joints)