Immunological techniques based on
primary antigen-antibody reactions
(2.)
7th week
practice
immunoblot
immunhistochemistry
flow cytometry
The sensitivity of immunoassays
Characterization of antigens by
Western blotting
Anode(+)
steps:
1. sample preparation (cells,
tissues)
2. gel electrophoresis
3. blotting
4. labeling
5. development
Cathode(-)
usage: identification of defined
components from protein mixtures by
antigen specific antibodies
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
‘Brute-force’ or ‘High throughput’
cytokine detection
(1.)
Cytokine array
Simultaneous detection of multiple cytokines
(the process is similar to the procedures of Western-blot after the blotting step)
multiple antigen specific antibodies bound to membrane
labeled antibody mixture
(+)
IL-2
IL-4
unknown cytokine
containing solution
…
…
MIP3β
IFN
…
(
-)
Disadvantage – defined volume sample needed to
cover the surface of the membrane
cytokine production of
moDC activated by CD40L and
CD40L+SLAM combination
Réthi és mtsi. 2006
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
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
Classical histochemistry
Acute bronchopneumonia (hematoxilin- eozin staining)
Only few cell types could be identified
Immunohistochemistry
(CD68+ macrophages and lymphocytes, granuloma)
Antinuclear (ANA) autoantiboies from the serum of a SLE patient can be visualized
in cell culture (Hep-2) by indirect fluorescent labeling (immunofluorescence)
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.
Flow cytometry
An immunofluorescent method that mutually
complements the fluorescent microscopy
• Investigation of different cells or particles travelling high
velocity in flow
• Detects fluorescence intensity and scattered light of the
labeled cells
• Can investigate enormous number of cells in short period
of time
Why flow cytometry
Most cells in the immune system can be found in free or loosely adherent
form. They can be easily suspensed and labeled by fluorescent antigen
specific antibodies, and then they can be examined cell by cell.
The cells’ light scatter and immunofluorescent properties can be analyzed
statistically (eg. percentages of different cell populations)
Rare cell populations can be identified and examined (eg. antigen specific
lymphocytes)
The method provide qualitative and quantitative data – it can detect the
presence of different antigens in the cell, and the expression levels of these
antigens. Changes in the expression of certain molecules can be followed
after different treatment of the specimen. (eg. cell activation, disease
progression)
Benchtop flow cytometer
Sorter - flow cytometer
(FACS station)
Example Chanel Layout for Laser-based Flow Cytometry
The emited fluorescent light can
be separeted to components by
special mirrors and filters
photodetectors
PMT 4
cell
suspension
in tube
flow cell
PMT 3
PMT 2
PMT 1
forward light scatter
detector
Laser
(PMT=photo-multiplayer tube)
The scattered light and multiple fluorescent color can be measured. It can be considered as a
fluorescent microscope with flow sample – but only with light intensity detection, no imaging
Light scatter and fluorescence
Forward angle light
scatter sensor
(FSC, FALS)
Laser
Can be loosely
considered as a
representation of the
particle size
Side light scatter(SSC) and fluorescence detectors
SSC represents the granularity of the cells
Multocolor staining can be used to identify cell sub-populations
(autofluorescence – presence of piridins and flavins)
Immunophenotyping
Example:
Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio
(eg. monitoring AIDS progression)
Labeling:
FITC labeled anti-CD4 antibody(α-CD4-FITC)
PE labeled anti-CD8 antibody (α-CD8-PE)
Th
NK
Tc
Lymphocytes in the periferial blood
Fluorescent
microscopy
B
high velocity flow stream
detecting CD4-FITC
labeled (TH) cell
(in cuvette or stream in air)
detector
signal
processing unit
CD8
PE
screen
increasing light
intensity
a dot representing a
CD4+ CD8- cell
CD4
FITC
microscopy:
detecting the PE labeled cell
(CD8-PE)
CD8
PE
detector
signal
processing unit
increasing light
intensity
CD4
FITC
detecting the unlabeled cell
(eg.B cell) by autofluorescence
CD8
PE
detector
Signal
processing unit
increasing light
intensity
microscopy:
dim (autofluorescent)
cell
CD4
FITC
CD8
PE
18%
44%
0%
quadrant
statistics
CD4 38%
FITC
Graphical representations 1.
dot-plot
contourplot
densityplot
Graphical representations 2.
Histogramm
Numeral intensity
values:
~7
~ 1300
homogenous cell
population is normally
distributed (Gaussian)
Different cell types - characteristic light scattering
granulocytes
side light scattering
(SSC)
(e.g. granulated)
monocytes
lymphocytes
forward light
scattering (FSC)
(„size”)
Examination of peripheral blood by haematology automats
Measured parameters:
peroxydase staining (the presence of myeloperoxydase, x – axis)
light scatter (high on large granular cells, y – axis)
1 Noise
2 Nucleated Red Blood Cells
3 Platelet Clumps
4 Lymphocytes and Basophils
5 Large Unstained Cells
6 Monocytes
7 Neutrophils
8 Eosinophils
Only the major cell types can be identified
Characterisation of immune cells using cell
surface markers
Cell types, differentiation stages can be identified using a
combination of cell surface markers.
Used in diagnostics:
- ratio of different cell types
- altered expression of cell surface markers
Examples:
- Inflammatory processes – increased neutrophil numbers
- HIV progression – decrease of CD4+ T cell count
CD4+ : CD8+ = 1.6
Normal CD4+ T cell count = 600 – 1400/l
AIDS = CD4+ T cell count <200/l
- increase of CD5+ B cells – typical for some B cell Leukemias
Diagnosis of immunodeficiency by flow cytometry
WAS: Wiscott-Aldrich Syndrome
A typical symptome:
Lacking or decreased CD43 expression
XLA: X-linked Agammaglobulinemia
Inhibited B cell development:
lack of CD19+ B cells
CD antigen
cell type
function
ligand
CD3
T cells
TCR signalling
-
CD4
helper T sejtek, (monocytes,
T cell coreceptor, (HIV
receptor)
MHC- II, HIV
pDC)
CD5
T cells, (B cell subset: B1)
adhesion, activation signals
CD72
CD8
cytotoxic T cells, (NK,  T cells)
T cell coreceptor
MHC I
CD14
monocytes, macrophages,
some granulocytes
LPS binding
LPS, LBP
CD19
B cells
part of CR2, B cell coreceptor
C3d, C3b
CD28
T cells
costimulatory signals to T
cells
(B7-1, B7-2)
CD80, CD86
CD34
hematopoietic progenitor cell
adhesion
CD62L
(L-selektin)
CD56
NK cell, (T and B cell subset)
homoadhesion (N-CAM
isoform)
APC: DC, B, monocyte,
macrophage
costimulatory signals
CD80, CD86
(B7-1, -2)
CD28, CD152
‘Brute-force’ or ‘High throughput’
cytokine detection
(2.)
Cytometric multiplex bead array (CBA)
Flow cytometric method with principles similar to cytokine array.
Cytokines are determined not by their positions on the membrane but
the fluorescent color intensities of the cytokine capture beads.
IL-2
cytokine array
CBA
IL-4
IL-8
…
…
IL-12
IL-23
IL-17
…
…
Cytometric Bead Array
The presence and the quantity of the cytokine can be
shown by a light intensity of the reporter antibody
labeled with different fluorescent color
reporter antibody
citokine
citokine capture
bead
Cytokine standards for evaluating the concentrations
IL-2
IL-4
…
…
Works with small volumes (5-20l), but requires special instruments
Cytometric bead array (CBA)
The principle of CBA is comparable with the sandwich ELISA
Sandwich ELISA
CBA
IL-2
IL-4
IL-2
IFN
Cytokine
quantity
TNFα IL-6 IL-8
IL-12 IL-10 IL-1β
500pg/ml
250pg/ml
125pg/ml
62pg/ml
31pg/ml
16pg/ml
Cytokine standard dilution series
8pg/ml
0
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 or CD150 (SLAM)
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/l);
- 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/l (phys.: 1000-1200/l);
- 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.
HIV supplemental confirmatory tests:
Western-blot:
The solid phase antigens are derived from LAV strain of HIV-1 grown in the CEM
cell line. You can obtain pre-wet blot membrane strips and incubate with the
examined serum. HIV specific serum antibodies result specific bands on the
Western blot. At least two envelop protein specific band or one envelop specific and the p24 antigen
specific band can be considered as positive test.
Immunofluorescent assay (IFA):
The assay uses immortalized human T-cells which express HIV-1 antigens on
their surface. The cells are fixed to the surface of an IFA glass slide. Fixed,
uninfected T-cells are provided as a control. The serum or plasma sample
HIV- I antibodies comes in contact with the HIV-1 antigens on the slide.
Fluorescent secondary antibodies detects them. The interpretation of the
degree and pattern of fluorescence of the infected cells of the IFA slide
compared to the uninfected cells determines the confirmed HIV-1 status of the
sample.
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.
Special cytometers
New fast computers and high speed imaging opened the possibility to record the
investigated cells’ images during flow cytometry.
Morphological data can be obtained by flow cytometry
Good tool to investigate some cellular function
(See on the coming pictures or the next seminars)
Imaging cytometry, LSC
(detailed morphology)
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