Radioimmunoassay, enzymoimmunoassay - principles, practical use.

Laboratory measurement of specific IgE antibodies.Laboratory measurement of autoantibodies.Flow cytometry - principles, practical use.Laboratory methods of assessment of cellular immunity.Laboratory methods of assessment of humoral immunity.

Humoral analytes

• Precipitation

• Aglutination

• Turbidimetry

• Nephelometry

Imunofixation of serum

Humoral analytes

• Precipitation

• Aglutination

• Turbidimetry

• Nephelometry

TURBIDIMETRIE

NEFELOMETRIE

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ELISA – assessment of specific antibodies

RAST

ELISA – assessment of antigen

Indirect immunofluorescence

Asessment of cellular imunity

• Number of cells - subpopulations

• Phagocytosis

• Activation of lymphocytes

Flow cytometry

Gating of lymphocytes

T and Th lymphocytes

T and B lymphocytes

Activated T lymphocytes

http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html

CD3+ T lymphocytes: mature T lymphocytes 50 - 75%

CD4+ T lymphocytes: helper T lymphocytes 30 - 60%

CD8+ T lymphocytes: cytoxic T lymphocytes 15 - 30%

CD25+ T lymphocytes: activated T lymphocytes 1 - 5%

CD19+ B lymphocytes: mature B lymphocytes 5 - 15%

CD56+ NK cells: natural killer cells 5 - 15%

CD14+ cells: monocytes

CD15+ cells: granulocytes

CD38+ cells: plasma cells

IMPORTANT LEUKOCYTE POPULATIONS

Ability to proliferateAbility to produce cytokines

TESTS OF PHAGOCYTIC FUNCTION

TESTS OF LYMPHOCYTE FUNCTION

No of granulocytesPhagocytar activity of granulocytesOxidative burst

Organ specific autoantibodies

• Anti-thyroidal AB

• Anti-pancreatic beta cells AB

• Anti-acetylcholine receptor AB

• Anti-gonadal AB

Systemic autoantibodies

• Anti-nuclear AB (ANA, ENA)

• Rheumatoid factor

• Anti-neutrophil cytoplasma AB (ANCA)

• Anti-phospholipid AB (APA)

Non-organ-specific autoantibodies in organ-localized autoimmune diseases

• ANCA (non-specific inflammatory bowel disease) //+ASCA//

• ATG (coeliac disease)

• SS-A, SS-B (Sjögren syndrome)