7th weekpractice

Immunological techniques based on primary antigen-antibody reactions

(2.)

immunoblot

immunhistochemistry

flow cytometry

The sensitivity of immunoassays

Characterization of antigens by Western blotting

steps:1. sample preparation (cells,

tissues)2. gel electrophoresis3. blotting4. labeling5. development

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

Anode(+)

Cathode(-)

• 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

StandardProtein sample

SDS-PAGE Membrane Western blot

Antibody recognizes epitope in specific protein

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

Fab

Fc

Epitope on protein surface

Primary antibody

Secondary antibody

Horseradish peroxidase

Immobilized proteins

Membrane

H2O2

H2O

luminol

enhancer

h

Detection

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)

luminol

‘Brute-force’ or ‘High throughput’ cytokine detection

(1.)

Cytokine arrayCytokine array(the process is similar to the procedures of Western-blot after the blotting step)

( - )

IFN

( + ) IL-2 IL-4 …

…

MIP3β …

multiple antigen specific antibodies bound to membrane

unknown cytokine containing solution

labeled antibody mixture

Simultaneous detection of multiple cytokines

Disadvantage – defined volume sample needed to cover the surface of the membrane

cytokine production ofmoDC activated by CD40L andCD40L+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)

Tissue sample

Fixation

Section before staining

Freezing

Sectioning

Immunohistochemistry

Immunohistochemistry ABC Method

Secondary antibody

Avidin

Cells

Slide

Primary antibody

Biotin

Enzim

X

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 mouseIgG (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 cytometryFlow 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 Can investigate enormous number of cells in short period of timeof 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 CytometryExample Chanel Layout for Laser-based Flow Cytometry

PMT 1

PMT 2

PMT 4

The emited fluorescent light can be separeted to components by

special mirrors and filters

Laser

PMT 3cell

suspension in tube

photodetectors

flow cell

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

Laser

Light scatter and fluorescence

Forward angle light scatter sensor(FSC, FALS)

Side light scatter(SSC) and fluorescence detectors

(autofluorescence – presence of piridins and flavins)

Can be loosely considered as a

representation of the particle size

SSC represents the granularity of the cells

Multocolor staining can be used to identify cell sub-populations

BNKTh Tc

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)

Lymphocytes in the periferial blood

Fluorescent microscopy

Immunophenotyping

focu

sed

lase

r bea

m

high velocity flow stream(in cuvette or stream in air)

CD4FITC

CD

8P

E

de

tec

tor

detecting CD4-FITC labeled (TH) cell

signal processing unit

screen

a dot representing aCD4+ CD8- cell

increasing light intensity

microscopy:

de

tec

tor

detecting the PE labeled cell(CD8-PE)

CD

8P

ECD4FITC

signal processing unit

increasing light intensity

de

tec

tor

detecting the unlabeled cell(eg.B cell) by autofluorescence

CD

8P

ECD4FITC

Signal processing unit

increasing light intensity

microscopy: dim (autofluorescent)

cell

quadrantstatistics

CD

8P

ECD4FITC

38%

0%

44%

18%

Graphical representations 1.

dot-plot

contour-plot

density-plot

Graphical representations 2.

Histogramm

homogenous cell population is normally distributed (Gaussian)

Numeral intensity values:

~ 7 ~ 1300

Different cell types - Different cell types - characteristic light scatteringcharacteristic light scattering

forward light scattering (FSC)

(„size”)

side light scattering (SSC)

(e.g. granulated)

granulocytes

monocytes

lymphocytes

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)

Only the major cell types can be identified

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

Characterisation of immune cells using cell Characterisation of immune cells using cell surface markerssurface 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+ = 1.6Normal 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

WAS: Wiscott-Aldrich Syndrome

XLA: X-linked Agammaglobulinemia

Inhibited B cell development: lack of CD19+ B cells

A typical symptome:Lacking or decreased CD43 expression

Diagnosis of immunodeficiency by flow cytometry

CD antigen cell type function ligand

CD3 T cells TCR signalling -

CD4 helper T sejtek, (monocytes, pDC)

T cell coreceptor, (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 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)

CD80, CD86 (B7-1, -2)

APC: DC, B, monocyte, macrophage

costimulatory signals CD28, CD152

‘Brute-force’ or ‘High throughput’ cytokine detection

(2.)

Cytometric multiplex bead array (CBA)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.

CBA cytokine array

IL-2

IL-4

IL-8

…

…

IL-12 IL-23 IL-17 … …

Cytometric Bead ArrayThe presence and the quantity of the cytokine can be shown by a light intensity of the reporter antibody labeled with different fluorescent color

IL-2IL-4……

Cytokine standards for evaluating the concentrations

citokine capture bead

citokine

reporter antibody

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

The principle of CBA is comparable with the sandwich ELISAThe principle of CBA is comparable with the sandwich ELISA

Sandwich ELISA CBA

Cytometric bead array (CBA)Cytometric bead array (CBA)

IL-2

IL-2

IL-4 IFN

IL-8IL-1β

IL-6IL-12 IL-10

TNFα

Cytokine quantity

Cytokine standard dilution series

500pg/ml 250pg/ml 125pg/ml 62pg/ml 31pg/ml 16pg/ml 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 deathand 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 runrampant and result in death. The mechanism responsible is now known to be thesupression 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 atwhich CD4+ cells are being destroyedexceeds the capacity of the host toreplenish them, their number decreasesto a point where cell-mediated immunity falters.

The failure of cell-mediatedimmunity renders to the host susceptible to fatal opportunisticinfections.

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 EXAMINED

PROTEINS

SAMPLE DETECTION

ELISAELISA Anti-HIV IgG and IgM antibodies

serum

(sometimes saliva

or urine)

with secondary (conjugate-) anti-IgG antibodies

Western blotWestern blot HIV capsid-proteins: p17, p24 , p6, p7

Envelop:

gp41, gp120, gp160;

serum with secondary (conjugate-) anti-IgG antibodies

After this finding they verified the parents:Both of them were HIV-positive.

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 mustbe verificated: usually a Western blot or another ELISA is performed.

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 complainedabout 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- andPseudomonas 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 respiratoryfailure, the lieutenant – in spite of his high serum HIV-antibody level – hasnot 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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