8th SEMINAR
LABORATORY METHODS BASED ON
ANTIGEN-ANTIBODY INTERACTIONS I
THE SENSITIVITY OF IMMUNOASSAYS
Sensitive methods:
• precise
• expensive
• usually used for verification
Less sensitive methods:
• give semiquantitative results
• cheap
• usually used for screening
IMMUNOAFFINITY
CHROMATOGRAPHY
Separation/purification of antigens or
antibodies from a mixture
AFFINITY PURIFICATION OF ANTIBODIES
USING AN ANTIGEN-SORBENT COLUMN
column
affinity purified antibody : monoclonal antibodies
which can be ordered from catalogues are also
purified using this technique
polymer beads
covalently bound
antigen
STEPS OF PURIFICATION
1)
2)
3)
4)
Addition of
antibodies to
be purified
Binding
Washing
Elution
PURIFICATION OF ANTIGENS
1) Loading the antigen mixture
2) Binding
column
3) Washing
4) Elution
polymer bead
fixed antigen-specific Abs on the surface of the bead
Purified antigens
ELISA
Enzyme Linked Immune Sorbent Assay
ELISA plate
well
Enzyme Linked
Immune Sorbent
enzyme
Antibody conjugated with
enzyme
Antigen/antibody
adsorbed to solid
surface
ENZYME ACTIVITY IN ELISA IS DIRECTLY
PROPORTIONAL TO THE AMOUNT OF
IMMUNECOMPLEX PRESENT
Enzyme activity is measured by the color
reaction due to conversion of substrate
Similar principle applies to many other antibody-based
detection methods
BASIC SETUPS IN
ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY
Direct method
Indirect method
Label
Label
Secondary
antibodies
Primary
antibodies
Antigen
BASIC SETUPS IN
ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY
Enzyme/anti-enzyme system
PAP – peroxidase / anti-peroxidase
APAAP – alkaline phosphatase / anti- alkaline phosphatase
Enzyme
Enzyme-specific
antibody, same isotype
as the primary antibody
Primary
antibody
Antigen
Secondary
antibody
BASIC SETUPS IN
ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY
Indirect systems combined with biotin-avidin signal amplification
(Avidin binds biotin with very high affinity )
Basic
Avidin-enzyme complexes
ABC
Avidin-biotin
enzyme
complexes
Biotin-enzyme complex
Avidin
Biotinylated antibody
Antigen
STEPS OF COMBINED/’SANDWICH’ ELISA
For antigens present at low concentration in complex biological samples
Coating with Ag-specific
„capture” antibody
Blocking free plastic
surface with inert protein
Removal of excess enzyme
Removal of unbound material
Removal of unbound protein
Removal of unbound material
Addition of antigencontaining solution
Addition of biotinylated
antibody specific to a different
epitope on target protein
Addition of avidinconjugated enzyme
Addition of substrate
STEPS OF BASIC INDIRECT ELISA
Detection of antigen or specific antibody
Removal of excess antigen
Removal of excess protein
Adsorption of antigen (coating)
Removal of excess antibody
Saturation of uncovered
surface area with proteins
Removal of excess antibody
Addition of Agspecific antibodies
Addition of Secondary Ab
conjugated with enzyme
Addition of chromogenic
substrate
EQUAL ABSORBANCE = EQUAL CONCENTRATION
OD
You should also dilute the
unknown sample
The sample with unknown
concentration
This region could indicate
the concentration
According to OD: it could be anyone
concentration
0.004
0.007
0.015
0.030
0.061
0.12
0.24
0.49
0.97
1.9
3.9
7.8
16
31
62
125
250
500
1000
?
0
PRACTICAL USE OF ELISA TECHNICS
Sandwich ELISA
Indirect ELISA
Measuring the amount of a given
antigen (molecule)
Measuring the amount of antigenspecific antibodies
• cytokines, hormones, drugs
• viral/bacterial antigens –
diagnosis of infection
• tumor antigens – diagnosis of
tumors / screening / follow-up
• pathogen-specific antibodies –
diagnosis of infection*
• isotype of antibodies – time
course, monoclonal antibodies
• autoantibodies – diagnosis of
autoimmune disorders
WESTERN BLOT
(IMMUNOBLOT)
Identification of defined components from
protein mixtures by antigen specific antibodies
Anode(+)
Steps:
1) Sample preparation (cells, tissues)
2) Gel electrophoresis
3) Blotting
4) Labeling (by primary and secondary antibodies)
5) Detection
Cathode(-)
WESTERN BLOT (IMMUNOBLOT)
Lysis of sample
Loading
Gel-electrophoresis
Standard
Protein
sample
SDS-PAGE
Blotting
Labeling
Primary antibody binds to its epitope
in the protein, then the labeled
secondary antibody binds to the
primary antibody
Membrane
X-ray film
IMMUNOPRECIPITATION
• Isolation and concentration of a
particular protein from a protein
mixture
• Detection of protein associations
(e.g. members of receptor
signalization)
CHROMATIN
IMMUNOPRECIPITATION
(ChIP)
Identification of molecules (mainly transcription
factors) binding to a specific site of the DNA
Provides information about the link between
signaling pathways and gene activation
IMMUNOHISTOCHEMISTRY
Labeled antibodies added to fixed tissue sections detect the
distribution of the chosen antigen within the tissue or within the cells
of a particular tissue
• Immunofluorescence
•Fluorescent dye coupled to antibody
FITC – fluorescein isothiocyanate (green)
PE – phycoerythrin (orange)
• Immunoenzyme method
• enzyme-coupled antibody
P – peroxidase
AP – alkaline phosphatase
(Substrates converted into an insoluble compound)
IMMUNOHISTOCHEMISTRY
Fixation
Tissue
sample
Freezing
Sectioning
Section before
staining
IMMUNOHISTOCHEMISTRY
Enzyme
Secondary antibody
Primary antibody
X
Avidin
Biotin
Slide
Cells
Tissue
sample
Classical histochemistry
Acute bronchopneumonia (hematoxylin-eozin staining)
Only few cell types could be identified
Immunohistochemistry
(CD68+ macrophages and lymphocytes, granuloma)
Antinuclear (ANA) autoantibodies from the serum of a SLE
patient can be visualized in cell culture (HEp-2) by indirect
fluorescent labeling (immunofluorescence)
A fixed and permeabilized skin fibroblast
Mitochondria
F-actin
Nucleus
Fixed and permeabilized pulmonary artery
endothelial cell
Peroxisomes
Mitochondria
Nuclei