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

zdroj světla kyveta s precipitátem detektor

zdroj světla

detektor

kyveta s precipitátem

rozptyl světla

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: monocytesCD15+ cells: granulocytesCD38+ 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)