7th week practice
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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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7th week practice

Immunological techniques based on primary antigen-antibody reactions

(2.)

7th weekpractice

  • immunoblot

  • immunhistochemistry

  • flow cytometry


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The sensitivity of immunoassays


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Characterization of antigens by

Western blotting

  • steps:

  • sample preparation (cells, tissues)

  • gel electrophoresis

  • blotting

  • labeling

  • development

Anode(+)

Cathode(-)

usage: identification of defined components from protein mixtures by antigen specific antibodies


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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 sample

Standard

Antibody recognizes epitope in specific protein

Western blot

Membrane

SDS-PAGE


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


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Enhanced chemiluminescence (ECL)

  • 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)

Immobilized proteins

Primary antibody

Fab

Fc

Horseradish peroxidase

luminol

H2O2

h

Epitope on protein surface

luminol

enhancer

Detection

H2O

Secondary antibody

Membrane


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‘Brute-force’ or ‘High throughput’

cytokine detection

(1.)


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Cytokine array

( + )

IL-2

IL-4

MIP3β

IFN

( - )

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


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cytokine production of

moDC activated by CD40L and

CD40L+SLAM combination

Réthi és mtsi. 2006


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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)


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Immunohistochemistry

Fixation

Sectioning

Tissue sample

Section before staining

Freezing


Immunohistochemistry abc method

ImmunohistochemistryABC Method

Enzim

Avidin

X

Biotin

Secondary antibody

Primary antibody

Slide

Cells

Tissue sample


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Classical histochemistry

Acute bronchopneumonia (hematoxilin- eozin staining)

Only few cell types could be identified


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Immunohistochemistry

(CD68+ macrophages and lymphocytes, granuloma)


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Antinuclear (ANA) autoantiboies from the serum of a SLE patient can be visualized in cell culture (Hep-2) by indirect fluorescent labeling (immunofluorescence)


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Immunohistochemistry using fluorescent detection

(TRITC)

Detection of actin microfilaments


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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-actinwaslabeled with green-fluorescent

Alexa Fluor 488 phalloidin (a mushroom toxin),and the nucleuswas stained with

TO-PRO-3 iodide


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Peroxisome labeling in fixed and permeabilized pulmonary artery

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.


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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


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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)


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Benchtop flow cytometer

Sorter - flow cytometer

(FACS station)


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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

PMT 3

flow cell

PMT 2

PMT 1

Laser

forward light scatter detector

(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


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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)


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Immunophenotyping

Fluorescent microscopy

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)

B

NK

Th

Tc

Lymphocytes in the periferial blood


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high velocity flow stream

(in cuvette or stream in air)

detecting CD4-FITC labeled (TH) cell

Th

increasing light intensity

a dot representing a

CD4+ CD8- cell

microscopy:

signal processing unit

screen

detector

CD8

PE

focused laser beam

CD4

FITC


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detecting thePElabeled cell

(CD8-PE)

Tc

increasing light intensity

signal processing unit

detector

CD8

PE

CD4

FITC


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detecting theunlabeledcell

(eg.B cell) by autofluorescence

B

increasing light intensity

microscopy:

dim (autofluorescent) cell

Signal processing unit

detector

CD8

PE

CD4

FITC


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0%

18%

CD8

PE

quadrant

statistics

38%

44%

CD4

FITC


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Graphical representations 1.

dot-plot

contour-

plot

density-

plot


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Graphical representations 2.

Histogramm

homogenous cell population is normally distributed (Gaussian)

Numeral intensity values:

~ 7

~ 1300


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Different cell types - characteristic light scattering

granulocytes

side light scattering (SSC)

(e.g.granulated)

monocytes

lymphocytes

forwardlight scattering (FSC)

(„size”)


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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 Noise2 Nucleated Red Blood Cells3 Platelet Clumps4 Lymphocytes and Basophils5 Large Unstained Cells6 Monocytes7 Neutrophils8 Eosinophils

Only the major cell types can be identified


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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


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Diagnosis of immunodeficiency by flow cytometry

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


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‘Brute-force’ or ‘High throughput’

cytokine detection

(2.)


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Cytometric multiplex bead array (CBA)

IL-2

IL-4

IL-8

IL-12

IL-23

IL-17

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.

cytokine array

CBA


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Cytometric Bead Array

IL-2

IL-4

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

Works with small volumes (5-20ml), but requires special instruments


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Cytometric bead array (CBA)

The principle of CBA is comparable with the sandwich ELISA

Sandwich ELISA

CBA

IL-2

IFNg

IL-4

IL-2


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Cytokine quantity

Cytokine standard dilution series

TNFα

IL-6

IL-8

IL-12

IL-10

IL-1β

500pg/ml

250pg/ml

125pg/ml

62pg/ml

31pg/ml

16pg/ml

8pg/ml

0


Acquired immune deficiency syndrome aids

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 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.


7th week practice

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.


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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;


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Oral candidiasis

esophageal candidiasis


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Diaper pimples


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  • 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;


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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.


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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.


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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.


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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)


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Imaging cytometry, LSC

(detailed morphology)

www.amnis.com


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