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ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGEN TS. ANTIGEN -ANTI BODY REA CTIONS WITH LABELED ANTI BODY. M. M - marker. A b. Fc. Fab. E NZYME - L INKED I MMUNO S ORBENT A SSAY (ELISA , EIA )- marker EN ZY M E. I MUNO F LUORESCEN CE (IF) - marker FLUORESCENT DYE.
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ANTIGEN-ANTIBODY REACTIONSWITHLABELED REAGENTS
ANTIGEN-ANTIBODY REACTIONS WITH LABELED ANTIBODY M M - marker Ab Fc Fab ENZYME-LINKEDIMMUNOSORBENTASSAY (ELISA, EIA)-marker ENZYME IMUNOFLUORESCENCE (IF) - marker FLUORESCENT DYE RADIOIMMUNOASSAY (RIA) - marker RADIOACTIVE ISOTOPE
ELISA Used for detection of antigen in liquide samples (e.g. serum) or antibodies (tipicallyin serum) Marker - enzyme, primary Ab (specificfor antigen) or secondary Ab (anti-Ig antibody) labeled Based on a change of colour ofthe substrateby an enzyme Signal detected by the spectrophotometer (absorbance measured) Bothqualitative andquantitaitve Performed in the plates for microtitration (96 well plate) enabling simultaneous analysis of large number of samples
ELISA Ab detection sample serum Ag –coated plate
ELISA Ab detection 12 6 Wash Incubation
E E E E E E ELISA Ab detection labelled secondary Ab
E E E E E E ELISA Ab detection 12 6 Incubation
E E E E E E E E ELISA Ab detection Wash
E E E E 12 ELISA Ab detection 6 Colorimetric detection
Ag ELISA (Ag detection in liquid sample - SANDWICH ELISA) sample (serum) unlabelled Ag-specific Ab
ELISA (Ag detection in liquid sample- SANDWICH ELISA) 12 6 Wash Incubation
E E E E E ELISA (Ag detection in liquid sample- SANDWICH ELISA) 12 labelled Ag-specific Ab 6 Wash Incubation
E E ELISA (Ag detection in liquid sample- SANDWICH ELISA) 12 adding supstrate 6 Colorimetric detection
ELISA (plate with positive samples)
Immunofluorescence (IF) Used for detectionof antigen in solid samples (e.g. on/in cells/tissues) or antibodies (tipically in serum) Marker - fluorochrome, primary Ab (specific for antigen) or secundary Ab (anti-Ig antibody) labeled Based on emission of visible light (colour) after exposure to a light of a defined wave length (UV light) (fluorescein –light green, rodamin – red ...) Signal detected using fluorescent microscope or flow cytometer Qualitative, semi-quantitative or quantitative
Detection of Agon/in cells/tissues DIRECT (DIF) (primary Ab labeled) IMMUNOFLUORESCENCE INDIRECT (IIF) (secondary Ab labeled)
F F F F DIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) Ag-specific labeled - Ab Fixed sample
F F F F DIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) 12 6 Washing Incubation
F F DIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) Excitation Fluorescence
Detection of Agon/in cells/tissues DIRECT (DIF) (primary Ab labeled) IMMUNOFLUORESCENCE Detection of Agon/in cells/tissues INDIRECT (IIF) (secondary Ab labeled)
INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) Ag-specific unlabeled Ab Fixed sample
INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) 6 Washing Incubation
F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) Secondary labeled Ab Incubation
F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) 12 6 Incubation
F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) Washing
F F F F INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) Excitation Fluorescence
Detection of Agon/in cells/tissues DIRECT (DIF) (primary Ab labeled) IMMUNOFLUORESCENCE Detection of Agon/in cells/tissues INDIRECT (IIF) (secondary Ab labeled) Detection of Abs in serum
INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum) Tested serum Fixed antigen
INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum) 6 Washing Incubation
F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum) Ig-specific labelled Ab Incubation
F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum) 12 6 Incubation
F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum) Ispiranje
F F F F INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum) Excitation Fluorescence
IIF ANCA (Anti-neutrophil cytoplasmic antibody) Neutrophils (pANCA) Perinuclear staining pattern Neutrophils (cANCA) Cytoplasmic staining pattern
IIF (Anti-glomerular basement membrane antibody) Glomerulonephritis Goodpasture’s syndrome
Ag detection DIRECT (DIF) (primary Ab fluorescently labeled) specificity IMUNOFLUORESCENCIJA Ag detection INDIRECT (IIF) (primary Ab fluorescently labeled) sensitivity Ab detection
Radio-imunotest (RIA) Nobelova nagrada za fiziologiju ili medicinu 1977 Rosalyn Yalow "for the development of radioimmunoassays of peptide hormones" Very sensitive quantitative test Used for measuring low quantities of Ag in a solution (e.g.blood hormone levels) Marker is radioactive isotope (e.g.125I) Bound radioactivity is measured by a counter Difficulties: radiation exposure during work and waste diposal
RAPID PREGNANCY TEST (principle) detection of beta HCG in urine of a pregnant women three antibodies used: 1. anti-HCG antibody – labeled with colloidal gold (free) 2. anti-HCG antibody – unlabeled (fixed on a membrane) 3. anti-Ig antibody – unlabeled (fixed on a membrane)
RAPID PREGNANCY TEST (results) + sample (urine) positive – sample (urine) negative
Z Z Z Z Z Z Z RAPID PREGNANCY TEST (positive result) sample (urine) positive sample (urine) unlabeled anti-HCG Ab anti-Ig Ab + HCG diffusion diffusion labelled anti-HCG Ab Z Z Z Z
Z Z Z Z Z RAPID PREGNANCY TEST (negative result) negative sample (urine) sample (urine) unlabeled anti-HCG Ab anti-Ig At – diffusion diffusion labeled anti-HCG Ab Z Z Z Z
1. ELISA is used for detection of a. enzyme 2. Antibody in ELISA is labeled by b. colorimeter 3. ELISA is done c. antigen or antibody in liqid samples 4. Substrate degraded by the enzyme in ELISA is detected by d. indirect imunofluorescenceor ELISA is used 5. In ELISA, the amount of antigen or antibody is measured by e. degraded substrate colour intensity 6. In indirect immunofluorescence, by fluorescent dye is labelled f. radioactive isotope 7. For detection ofChlamydiain cervical swab is used g. 96 well - plate 8. For detection of auto-antibodies in serum h. RIA 9. RIA as a marker utilises i. direct imunofluorescence 10. For blood hormone level determination is used j. anti-imunoglobuline antibody c a g b e j i d f h