Analytical and preparative methods
based on primary
antigen-antibody binding
Immunoaffinity chromatography,
ELISA,
immunoblot,
immunohistochemistry
Affinity purification of antibodies using an antigen-sorbent column
column
polymer beads
Column content
Covalently bound
antigen
Addition of
antibodies to
be purified
Binding
Washing
elution
Antigen-specific
polyclonal antibodies
The reverse setup is also used
Purification of antigen on antibody-immunosorbent column
polymer beads
column
Loading the antigen mixture
Binding
Wash
Column content
Elution
Covalently bound
antibodies
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 antigen 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
Indirect method
Direct 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
Examples of direct, indirect and competitive
ELISA systems
Steps of the basic indirect ELISA
Detection of antigen or specific antibody
Adsorption of antigen (coating) Removal of excess antibody
Saturation of
uncovered surface
area with protein such
as BSA, casein etc..
Addition of Agspecific antibodies
Addition of
Secondary Ab
conjugated with
enzyme
Addition of
chromogenic
substrate
Removal of excess antigen
Removal of excess protein
Steps of the combined sandwich ELISA
For antigens present at low concentration in complex biological samples
Coating with Agspecific „capture”
antibody
Blocking free plastic
surface with inert
protein
Addition of antigencontaining solution
Addition of biotinylated
antibody specific to a
different epitope on
target protein
Addition of avidinconjugated enzyme
Addition of substrate
Removal of excess enzyme
Removal of unbound material
Removal of unbound protein
Removal of unbound material
Competitive ELISA
Highly sensitive method used to detect and quantitate small
amounts of antigen in complex biological samples. Antigen in
solution and on the solid surface compete for the binding site of
labeled specific antibody.
- Coat with antigen, blocking
- Add experimental sample that contains or lacks antigen
-Add labeled antibody, --- Binding
- Washes
- Add substrate
ELISA
Background due to non-specific binding to plastic
Labeled antibody bound to
plastic directly or indirectly gives
the same signal as those that
bind specifically
Nonspecific binding can be
controlled by:
•Saturation of free plastic
surfaces
•Using detergents
ELISA plates - results
The chromogens used for detection are
CARCINOGENOUS!
Characterization of antigens by
Western blotting
Western Blot
• The use of antibodies in molecular biology is widespread
• It is probably most often encountered in Western analysis
• SDS-PAGE gel  resolved into single protein bands (overlap possible)
• Presence of a protein is determined by hybridizing the proteins,
transferred or applied to a membrane, with the relevant antibody
Protein
Standard sample
Antibody recognizes
epitope in specific protein
SDS-PAGE
Membrane
Western blot
Western Blot
• Used to detect specific proteins
in a sample
• Proteins separated by Sodium
Dodecyl Sulfate-Polyacrylamide
Gel Electrophoresis (SDSPAGE), transferred to a
membrane
• Primary (1st) antibody
(monoclonal or polyclonal) used
to detect protein
• Enzyme linked 2nd antibody (e.g.
horseradish peroxidase-linked)
used to detect 1st antibody
Enhanced chemiluminescence (ECL)
Immobilized
proteins
Primary
Fabantibody
Fc
•In the presence of H2O2, horseradish peroxidase (HRP)
oxidizes diacylhydrazides such as luminol
• Directly after oxidation, luminol is in an exited state, and
emits a photon to return to the ground state
• This photon can be detected with a film or a camera
• Light emission can be enhanced by ~1000-fold with
phenolic compounds such as 6-hydroxybenzothiazole
(enhancer)
Horseradish
peroxidase
H2O2
Epitope on
protein
surface
Secondary
antibody
Membrane
H2O
luminol
luminol
h
enhancer
Detection
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
PE – phycoerythrin
• Immunoenzyme method
• enzyme-coupled antibody
P – peroxidase
PA – alkaline phosphatase
(Substrates converted into an insoluble compound)
Immunohistochemistry
Fixation
Tissue
sample
Freezing
Sectioning
Section before
staining
Immunohistochemistry
ABC Method
Enzim
X
Avidin
Biotin
Secondary antibody
Primary antibody
Slide
Cells
Tissue
sample
Histochemistry
Acute bronchopneumonia (hematoxilin- eozin staining)
Immunohistochemistry
(CD68+ macrophages and lymphocytes, granuloma)
Immunohistochemistry using fluorescent detection
Anti-nuclear autoantibodies in SLE (FITC)
Immunohistochemistry using fluorescent detection
(TRITC)
Detection of actin microfilaments
A fixed and permeabilized skin fibroblast. Mitochondria were labeled with mouse
IgG (anti–OxPhos Complex V) and visualized using goat anti–mouse IgG conjugated
with orange-fluorescent Alexa Fluor 555. F-actin was labeled with green-fluorescent
Alexa Fluor 488 phalloidin (a mushroom toxin), and the nucleus was stained with
TO-PRO-3 iodide
Peroxisome labeling in fixed and permeabilized pulmonary artery
endothelial cell. Peroxisomes were labeled using an antibody directed at
peroxisomal membrane protein 70 and detected with Alexa Fluor 488–labeled
goat anti–mouse IgG. Mitochondria were stained with MitoTracker Red prior
to fixation; nuclei were stained with blue-fluorescent DAPI.
Acquired Immune Deficiency
Syndrome – AIDS
Certain infectious microorganisms can supress or subvert the immune system.
At the beginning of the last century, when tuberculosis was the leading cause of death
and fully half the population was tuberculin-positive, it was well-known that an intercurrent measles infection would cause a well-contained tuberculosis infection to run
rampant and result in death. The mechanism responsible is now known to be the
supression of IL-2 synthesis after binding of measles virus to CD46 on macrophages.
Some of the microorganisms that supress immunity act by infecting
lymphocytes.
The human immunodeficiency virus (HIV) presents a chilling example of the
consequences of infection and destruction of immune cells by a microorganism.
The T-cell surface CD4 molecule acts as a receptor for HIV. CD4 is also expressed
on the surface of cells of the macrophage lineage and they too can be infected by this virus.
The clinical latency is long,
usually it means several years.
During this period, the level of CD4+
cells and virus particles in the blood
changes. When the rate at
which CD4+ cells are being destroyed
exceeds the capacity of the host to
replenish them, their number decreases
to a point where cell-mediated
immunity falters.
The failure of cell-mediated
immunity renders to the host
susceptible to fatal opportunistic
infections.
Case study:
The Pinkerton-family: infected blood caused tragedy
Benjamin Pinkerton was a US-navy lieutenant who saw service at Japan. He married with a
japanese woman during his service, who gave birth two healthy girls in 1987. She bore a boy
four years later, who seemed healthy, as well. The boy got the routine DPT-vaccination and an
oral polio-virus immunization.
These vaccinations had no side-effect and the boy grew normally.
At the age of six months he got sick and started to lose weight. He had severe, chronic diarrhea
with fever. Besides a chronic oral candidiasis, the boy got two otitis, one after the other .
The navy doctors examined the baby several times and prescribed antibiotic but it proved
ineffectual.
Results of somatic examination:
- body-temperature 38oC;
- candidiasis on the lateral sides of tongue
and on the mucosal surface of the oral cavity;
- „diaper-pimples”, which is also caused by Candida
infection;
- at respiration a subtle,
slurping noise was heard in each pulmonary lobes;
Oral candidiasis
esophageal candidiasis
Diaper pimples
Laboratory:
- normal amount of leukocytes (6500/ml);
- normal rate of leukocytes (neutrophyl 62%;
lymphocyte 30%; monocyte 5%; eosinophyl 2%; basophyl 1%);
- normal serum immunoglobulin levels:
- serum IgG: 997 mg/dl (phys.: 800-1000 mg/dl);
- serum IgM: 73 mg/dl (phys.: 50-150 mg/dl);
- IgA: 187 mg/dl (phys.: 150-300 mg/dl);
- normal amount of CD8 + T-cells, but the rate of CD4+ T-cells
is very low, only 85/ml (phys.: 1000-1200/ml);
- intradermal Candida-antigen did not evoke late-type
hypersensitvity reaction;
- results of ELISA and Western-blot analysis:
HIV-antibodies in the serum;
METHOD
ELISA
Western blot
EXAMINED
PROTEINS
SAMPLE
DETECTION
Anti-HIV IgG and
IgM antibodies
serum
(sometimes saliva
or urine)
with secondary
(conjugate-) anti-IgG
antibodies
HIV capsid-proteins:
p17, p24 , p6, p7
Envelop:
gp41, gp120, gp160;
serum
with secondary
(conjugate-) anti-IgG
antibodies
During HIV-diagnostics samples are always analyzed firstly by ELISA-method.
In case of reactivity (serum positive) two more measurements are needed
(2nd and 3rd analysis).
If the 2nd and 3rd measurements show reactivity as well, the subject’s sample must
be verificated: usually a Western blot or another ELISA is performed.
After this finding they verified the parents:
Both of them were HIV-positive.
While Pinkerton was healthy, his wife was feeling unwell and complained
about the swelling of her cervical lymphatic nodes.
It turned out, that she was pregnant right before the boy’s birth. At the end of pregnancy
the fetus had died and had to be removed by caesarean section.
The operation was going well but – because of the loss of blood –
she needed blood-transfusion (she got two units of blood).
The boy got two severe infections in turn: Pneumocystis carinii- and
Pseudomonas aeruginosa. He had serious cough with bloody spit (hemoptysis).
A week after this he died.
The parents got AZT- (zidovudin) therapy. While his wife died soon in respiratory
failure, the lieutenant – in spite of his high serum HIV-antibody level – has
not had symptoms yet.