ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS

ANTIGEN-ANTIBODY REACTIONS WITH LABELED ANTIBODY M Fc Fab E NZYME- L INKED I MMUNO S ORBENT A SSAY (ELISA, EIA)-marker ENZYME I MUNO F LUORESCENCE (IF) - marker FLUORESCENT DYE R ADIO I MMUNO A SSAY (RIA) - marker RADIOACTIVE ISOTOPE AbAb M - marker

ELISA Marker - enzyme, primary Ab (specific for antigen) or secondary Ab (anti-Ig antibody) labeled Performed in the plates for microtitration (96 well plate) enabling simultaneous analysis of large number of samples Signal detected by the spectrophotometer (absorbance measured) Used for detection of antigen in liquide samples (e.g. serum) or antibodies (tipically in serum) Based on a change of colour of the substrate by an enzyme Both qualitative and quantitaitve

ELISA Ab detection Ag –coated plate sample serum

Incubation Wash 6 12 ELISA Ab detection

labelled secondary Ab E E E E E E ELISA Ab detection

Incubation EEEE E E 12 6 ELISA Ab detection

Wash EEEE E E E E ELISA Ab detection

EEE E 12 6 Colorimetric detection ELISA Ab detection

ELISA (Ag detection in liquid sample - SANDWICH ELISA) unlabelled Ag-specific Ab sample (serum) Ag

Incubation 12 6 Wash ELISA (Ag detection in liquid sample - SANDWICH ELISA)

Incubation 12 6 Wash EEEEE labelled Ag-specific Ab ELISA (Ag detection in liquid sample - SANDWICH ELISA)

Colorimetric detection 12 6 EE adding supstrate ELISA (Ag detection in liquid sample - SANDWICH ELISA)

ELISA (plate with positive samples)

Immunofluorescence (IF) Marker - fluorochrome, primary Ab (specific for antigen) or secundary Ab (anti-Ig antibody) labeled Signal detected using fluorescent microscope or flow cytometer Used for detection of antigen in solid samples (e.g. on/in cells/tissues) or antibodies (tipically in serum) Based on emission of visible light (colour) after exposure to a light of a defined wave length (UV light) (fluorescein –light green, rodamin – red...) Qualitative, semi-quantitative or quantitative

IMMUNOFLUORESCENCE INDIRECT (IIF) (secondary Ab labeled) DIRECT (DIF) (primary Ab labeled) Detection of Ag on/in cells/tissues

DIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) F F F F Fixed sample Ag-specific labeled - Ab

F FF F Incubation Washing 12 6 DIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)

F F Excitation Fluorescence DIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)

DIF Chlamydia trachomatis

IMMUNOFLUORESCENCE INDIRECT (IIF) (secondary Ab labeled) DIRECT (DIF) (primary Ab labeled) Detection of Ag on/in cells/tissues

Ag-specific unlabeled Ab INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) Fixed sample

Incubation Washing 6 INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)

Incubation Secondary labeled Ab F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)

Incubation FFFFF F F F 12 6 INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)

Washing FFFFF F F F INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)

FFF F Excitation Fluorescence INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)

IMMUNOFLUORESCENCE INDIRECT (IIF) (secondary Ab labeled) DIRECT (DIF) (primary Ab labeled) Detection of Ag on/in cells/tissues Detection of Abs in serum Detection of Ag on/in cells/tissues

Fixed antigen Tested serum INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)

Incubation Washing 6 INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)

Incubation Ig-specific labelled Ab F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)

Incubation FFFFF F F F 12 6 INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)

Ispiranje FFFFF F F F INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)

FFF F Excitation Fluorescence INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)

IIF Neutrophils (cANCA) Cytoplasmic staining pattern ANCA (Anti-neutrophil cytoplasmic antibody) Neutrophils (pANCA) Perinuclear staining pattern

IIF Glomerulonephritis Goodpasture’s syndrome (Anti-glomerular basement membrane antibody)

IMUNOFLUORESCENCIJA INDIRECT (IIF) (primary Ab fluorescently labeled) DIRECT (DIF) (primary Ab fluorescently labeled) Ag detection Ab detection specificity sensitivity

Radio-imunotest (RIA) Marker is radioactive isotope (e.g. 125 I) Difficulties: radiation exposure during work and waste diposal Used for measuring low quantities of Ag in a solution (e.g. blood hormone levels) Very sensitive quantitative test Bound radioactivity is measured by a counter "for the development of radioimmunoassays of peptide hormones" Rosalyn Yalow Nobelova nagrada za fiziologiju ili medicinu 1977

Rapid pregnancy test (demonstration)

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)

positive sample (urine) negative sample (urine) + – RAPID PREGNANCY TEST (results)

positive sample (urine) Z Z Z Z diffusion Z Z Z HCG labelled anti-HCG Ab unlabeled anti-HCG Ab anti-Ig Ab + sample (urine) Z Z RAPID PREGNANCY TEST (positive result) Z Z

negative Z Z Z Z diffusion Z Z Z Z Z – RAPID PREGNANCY TEST (negative result) sample (urine) sample (urine) unlabeled anti-HCG Ab anti-Ig At labeled anti-HCG Ab

anti-imunoglobuline antibodyj.For blood hormone level determination is used 10. direct imunofluorescencei.RIA as a marker utilises9. RIAh.For detection of auto-antibodies in serum well - plateg.For detection of Chlamydia in cervical swab is used 7. radioactive isotopef.In indirect immunofluorescence, by fluorescent dye is labelled 6. degraded substrate colour intensitye.In ELISA, the amount of antigen or antibody is measured by 5. indirect imunofluorescence or ELISA is used d.Substrate degraded by the enzyme in ELISA is detected by 4. antigen or antibody in liqid samplesc.ELISA is done3. colorimeterb.Antibody in ELISA is labeled by2. enzymea.ELISA is used for detection of1. 1.____2.____3.____4.____5.____6.____7.____8.____9.____10.____ cagbejidfh