antigen-antibody reactions with labeled reagents
TRANSCRIPT
![Page 1: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/1.jpg)
ANTIGEN-ANTIBODYREACTIONS WITH LABELED
REAGENTS
![Page 2: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/2.jpg)
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
![Page 3: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/3.jpg)
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
![Page 4: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/4.jpg)
ELISA Ab detection
Ag –coated plate
sample serum
![Page 5: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/5.jpg)
IncubationWash
6
12
ELISA Ab detection
![Page 6: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/6.jpg)
labelledsecondary Ab
EE
E
E
E
E
ELISA Ab detection
![Page 7: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/7.jpg)
Incubation
EE
E
E
E
E
12
6
ELISA Ab detection
![Page 8: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/8.jpg)
Wash
EE
E E
EE
E
E
ELISA Ab detection
![Page 9: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/9.jpg)
E
E E
E
12
6
Colorimetric detection
ELISA Ab detection
![Page 10: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/10.jpg)
ELISA (Ag detection in liquid sample - SANDWICH ELISA)
unlabelledAg-specificAb
sample(serum)Ag
![Page 11: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/11.jpg)
Incubation
12
6
Wash
ELISA (Ag detection in liquid sample - SANDWICH ELISA)
![Page 12: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/12.jpg)
Incubation
12
6
Wash
E
EE
E
E
labelledAg-specificAb
ELISA (Ag detection in liquid sample - SANDWICH ELISA)
![Page 13: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/13.jpg)
Colorimetric detection
12
6 E E
adding supstrate
ELISA (Ag detection in liquid sample - SANDWICH ELISA)
![Page 14: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/14.jpg)
ELISA(plate with positive samples)
![Page 15: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/15.jpg)
![Page 16: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/16.jpg)
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
![Page 17: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/17.jpg)
IMMUNOFLUORESCENCE
INDIRECT (IIF)(secondary Ab labeled)
DIRECT (DIF)(primary Ab labeled)
Detection of Ag on/in cells/tissues
![Page 18: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/18.jpg)
DIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
F
FF
F
Fixed sample
Ag-specificlabeled - Ab
![Page 19: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/19.jpg)
F
FF
F
IncubationWashing
12
6
DIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
![Page 20: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/20.jpg)
F F
Excitation
Fluorescence
DIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
![Page 21: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/21.jpg)
DIF Chlamydia trachomatis
![Page 22: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/22.jpg)
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
![Page 23: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/23.jpg)
Ag-specificunlabeled Ab
INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
Fixed sample
![Page 24: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/24.jpg)
IncubationWashing
6
INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
![Page 25: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/25.jpg)
Incubation
Secondarylabeled AbF
F
F
F
F
FF
F
INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
![Page 26: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/26.jpg)
Incubation
FF
F
F
F
FF
F
12
6
INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
![Page 27: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/27.jpg)
Washing
FF
F F
FF
F
F
INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
![Page 28: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/28.jpg)
F
F F
F
Excitation
Fluorescence
INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample)
![Page 29: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/29.jpg)
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
![Page 30: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/30.jpg)
Fixedantigen
Tested serum
INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)
![Page 31: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/31.jpg)
IncubationWashing
6
INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)
![Page 32: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/32.jpg)
Incubation
Ig-specificlabelled AbF
F
F
F
F
FF
F
INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)
![Page 33: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/33.jpg)
Incubation
FF
F
F
F
FF
F
12
6
INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)
![Page 34: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/34.jpg)
Ispiranje
FF
F F
FF
F
F
INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)
![Page 35: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/35.jpg)
F
F F
F
Excitation
Fluorescence
INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum)
![Page 36: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/36.jpg)
IIF
Neutrophils (cANCA)
Cytoplasmic staining pattern
ANCA (Anti-neutrophil cytoplasmic antibody)
Neutrophils (pANCA)
Perinuclear staining pattern
![Page 37: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/37.jpg)
IIF
Glomerulonephritis Goodpasture’s syndrome
(Anti-glomerular basement membrane antibody)
![Page 38: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/38.jpg)
IMUNOFLUORESCENCIJA
INDIRECT (IIF)(primary Ab fluorescently labeled)
DIRECT (DIF)(primary Ab fluorescently labeled)
Ag detection
Ag detection
Ab detection
specificity
sensitivity
![Page 39: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/39.jpg)
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
![Page 40: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/40.jpg)
Rapid pregnancy test(demonstration)
![Page 41: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/41.jpg)
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)
![Page 42: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/42.jpg)
positive
sample (urine)
negative
sample (urine)
+
–
RAPID PREGNANCY TEST (results)
![Page 43: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/43.jpg)
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
![Page 44: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/44.jpg)
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
![Page 45: ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGENTS](https://reader035.vdocuments.site/reader035/viewer/2022062423/56649ea15503460f94ba4d10/html5/thumbnails/45.jpg)
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