ANALYTICAL AND PREPARATIVE
METHODS BASED ON PRIMARY
ANTIGEN-ANTIBODY BINDING (2.)
immunoblot (Western blot)
immunhistochemistry
flow cytometry
THE SENSITIVITY OF IMMUNOASSAYS
WESTERN BLOT
Steps:
Anode(+)
1) sample preparation
(cells, tissues)
2) gel electrophoresis
3) blotting
4) labeling
Cathode(-)
5) detection
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
Antibody recognizes
epitope in specific protein
Protein
Standard sample
SDS-PAGE
Membrane
Western blot
WESTERN BLOT
Used to detect specific proteins in a
sample
Proteins separated by Sodium
Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis (SDS-PAGE),
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
WESTERN BLOT
Detection methods:
Colorimetric (peroxidase enzyme – densitometry /
spectrophotometry)
Chemiluminescent (ECL)
Radioactive (isotope – X-ray)
Fluorescent (staining – CCD camera)
ENHANCED CHEMILUMINESCENCE (ECL)
•
•
Immobilized
proteins
•
•
Primary
Fabantibody
Fc
Horseradish
peroxidase
H2O2
Epitope on
protein
surface
Secondary
antibody
Membrane
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)
H2O
luminol
luminol
h
enhancer
Detection
CYTOKINE ARRAY
Simultaneous detection of multiple cytokines
multiple antigen specific antibodies bound to membrane
labeled antibody mixture
(+)
IL-2
IL-4
unknown cytokine
containing solution
…
…
MIP3β
IFN
…
(
Disadvantage: Relatively large sample volume is
needed to cover the surface of the membrane
-)
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
AP – alkaline phosphatase
(Substrates converted into an insoluble compound)
IMMUNOHISTOCHEMISTRY
Fixation
Tissue
sample
Freezing
Sectioning
Section before
staining
IMMUNOHISTOCHEMISTRY
Enzyme
X
Avidin
Biotin
Secondary antibody
Primary antibody
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)
Detection of actin microfilaments (TRITC)
A fixed and permeabilized skin fibroblast
Mitochondria
F-actin
Nucleus
Fixed and permeabilized pulmonary artery endothelial cell
Peroxisomes
Mitochondria
Nuclei
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
ADVANTAGES OF 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 (e.g. percentages of different cell populations)
Rare cell populations can be identified and examined (e.g. 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.
(e.g. cell activation, disease progression)
Benchtop flow cytometer
Sorter - flow cytometer
(FACS station)
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
Multicolor staining can be used to identify cell sub-populations
(autofluorescence – presence of piridins and flavins)
IMMUNOPHENOTYPING BY FACS
Example:
Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio
(e.g. 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 peripheral blood sample
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
(e.g. 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
CHARACTERIZATION 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 signaling
-
CD4
helper T cells, (monocytes, pDC)
T cell co-receptor, (HIV
receptor)
MHC- II, HIV
CD5
T cells, (B cell subset: B1)
adhesion, activation signals
CD72
CD8
cytotoxic T cells, (NK,  T cells)
T cell co-receptor
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
co-stimulatory signals to T
cells
(B7-1, B7-2)
CD80, CD86
CD34
hematopoietic progenitor cell
adhesion
CD62L
(L-selectin)
CD56
NK cell, (T and B cell subset)
homoadhesion (N-CAM
isoform)
APC: DC, B, monocyte,
macrophage
co-stimulatory signals
CD80, CD86
(B7-1, -2)
CD28, CD152
ACQUIRED IMMUNE DEFICIENCY
SYNDROME – AIDS
Certain infectious microorganisms can suppress 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 suppression of IL-2 synthesis after binding of measles virus to
CD46 or CD150 (SLAM) on macrophages and dendritic cells.
Some of the microorganisms that suppress 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 (neutrophil 62%; lymphocyte 30%;
monocyte 5%; eosinophil 2%; basophil 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
Anti-HIV IgG and
IgM antibodies
serum
HIV capsid-proteins:
p17, p24 , p6, p7
Envelop:
gp41, gp120, gp160;
serum
(sometimes saliva
or urine)
DETECTION
with secondary
(conjugate-) anti-IgG
antibodies
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 verified: 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.
highly active antiretroviral therapy – HAART
2 RT inhibitors + 1 protease inhibitor