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. steps: sample preparation (cells, tissues) gel electrophoresis
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The sensitivity of immunoassays reactions
Characterization of antigens by reactions
usage: identification of defined components from protein mixtures by antigen specific antibodies
Western Blot reactions
Antibody recognizes epitope in specific protein
Enhanced chemiluminescence (ECL) reactions
Epitope on protein surface
‘ reactionsBrute-force’ or ‘High throughput’
Cytokine array reactions
( + )
( - )
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
unknown cytokine containing solution
Disadvantage – defined volume sample needed to cover the surface of the membrane
cytokine production of reactions
moDC activated by CD40L and
Réthi és mtsi. 2006
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
Section before staining
Classical histochemistry reactions
Acute bronchopneumonia (hematoxilin- eozin staining)
Only few cell types could be identified
(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 patient can be visualized in cell culture
Detection of actin microfilaments
A fixed and permeabilized skin fibroblast. patient can be visualized in cell culture 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-actinwaslabeled with green-fluorescent
Alexa Fluor 488 phalloidin (a mushroom toxin),and the nucleuswas stained with
endothelial cell.Peroxisomeswere 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 artery
An immunofluorescent method that mutually complements the fluorescent microscopy
Why flow cytometry artery
Benchtop flow cytometer artery
Sorter - flow cytometer
The emited fluorescent light can be separeted to components by special mirrors and filters
cell suspension in tube
forward light scatter detector
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 f artery luorescence
Forward angle light scatter sensor
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)
Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio
(eg. monitoring AIDS progression)
FITC labeled anti-CD4 antibody(α-CD4-FITC)
PE labeled anti-CD8 antibody (α-CD8-PE)
Lymphocytes in the periferial blood
high velocity flow stream artery
(in cuvette or stream in air)
detecting CD4-FITC labeled (TH) cell
increasing light intensity
a dot representing a
CD4+ CD8- cell
signal processing unit
focused laser beam
detecting the artery PElabeled cell
increasing light intensity
signal processing unit
detecting the artery unlabeledcell
(eg.B cell) by autofluorescence
increasing light intensity
dim (autofluorescent) cell
Signal processing unit
Graphical representations artery 1.
Graphical representations artery 2.
homogenous cell population is normally distributed (Gaussian)
Numeral intensity values:
Different cell types - artery characteristic light scattering
side light scattering (SSC)
forwardlight scattering (FSC)
peroxydase staining (the presence of myeloperoxydase, x – axis)
light scatter (high on large granular cells, y – axis)
1 Noise2 Nucleated Red Blood Cells3 Platelet Clumps4 Lymphocytes and Basophils5 Large Unstained Cells6 Monocytes7 Neutrophils8 Eosinophils
Only the major cell types can be identified
Cell types, differentiation stages can be identified using a combination of cell surface markers.
WAS: Wiscott-Aldrich Syndrome
XLA: X-linked Agammaglobulinemia
A typical symptome:
Lacking or decreased CD43 expression
Inhibited B cell development:
lack of CD19+ B cells
‘ artery Brute-force’ or ‘High throughput’
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.
Cytometric Bead Array artery
The presence and the quantity of the cytokine can be shown by a light intensity of the reporter antibody labeled with different fluorescent color
citokine capture bead
Cytokine standards for evaluating the concentrations
Works with small volumes (5-20ml), but requires special instruments
Cytometric artery bead array (CBA)
The principle of CBA is comparable with the sandwich ELISA
Cytokine quantity artery
Cytokine standard dilution series
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 inter-
current 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
The failure of cell-mediated
immunity renders to the host
susceptible to fatal opportunistic
Case study: infecting
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
Oral candidiasis infecting
Diaper pimples infecting
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.
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.
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 complained
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 complained