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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. - PowerPoint PPT Presentation
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ANTIGEN-ANTIBODYREACTIONS WITH LABELED
REAGENTS
ANTIGEN-ANTIBODY REACTIONS WITH LABELED ANTIBODY
M
Fc
Fab
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA, EIA)-marker ENZYME
IMUNOFLUORESCENCE (IF) - marker FLUORESCENT DYE
RADIOIMMUNO ASSAY (RIA) - marker RADIOACTIVE ISOTOPE
AbM - 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
IncubationWash
6
12
ELISA Ab detection
labelledsecondary Ab
EE
E
E
E
E
ELISA Ab detection
Incubation
EE
E
E
E
E
12
6
ELISA Ab detection
Wash
EE
E E
EE
E
E
ELISA Ab detection
E
E E
E
12
6
Colorimetric detection
ELISA Ab detection
ELISA (Ag detection in liquid sample - SANDWICH ELISA)
unlabelledAg-specificAb
sample(serum)Ag
Incubation
12
6
Wash
ELISA (Ag detection in liquid sample - SANDWICH ELISA)
Incubation
12
6
Wash
E
EE
E
E
labelledAg-specificAb
ELISA (Ag detection in liquid sample - SANDWICH ELISA)
Colorimetric detection
12
6 E E
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
FF
F
Fixed sample
Ag-specificlabeled - Ab
F
FF
F
IncubationWashing
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
Detection of Ag on/in cells/tissues
Ag-specificunlabeled Ab
INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
Fixed sample
IncubationWashing
6
INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
Incubation
Secondarylabeled AbF
F
F
F
F
FF
F
INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
Incubation
FF
F
F
F
FF
F
12
6
INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
Washing
FF
F F
FF
F
F
INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
F
F F
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
Fixedantigen
Tested serum
INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)
IncubationWashing
6
INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)
Incubation
Ig-specificlabelled AbF
F
F
F
F
FF
F
INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)
Incubation
FF
F
F
F
FF
F
12
6
INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)
Ispiranje
FF
F F
FF
F
F
INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)
F
F F
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
Ag detection
Ab detection
specificity
sensitivity
Radio-imunotest (RIA)
Marker is radioactive isotope (e.g. 125I)
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
diffusion
Z
HCG
labelledanti-HCG
Ab
unlabeledanti-HCG
Ab
anti-IgAb
+
sample(urine)
Z
Z
RAPID PREGNANCY TEST (positive result)
Z Z
negative
Z Z ZZ
diffusion Z Z
diffusion
ZZ Z–
RAPID PREGNANCY TEST (negative result)
sample (urine)
sample(urine) unlabeled
anti-HCGAb
anti-IgAt
labeledanti-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
8.
96 well - plate g.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.____c a g b e j i d f h