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