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Labelling_Conjugated

Monoclonal & Polyclonal


Polyclonal

Monoclonal

Contains many antibodies


recognizing many
detedminants on an antigen

Contains a single antibody


recognizing only a single
determination

Various classes of
antibodies are present
(IgG,IgM,and so on)

Single class of antibody


produced

Can make a specific


antibody using only
a highly purified antigen

Can make a specific


antibody using an
impure antigen

Reproducibility and
standardization difficult

Highly reproducible

Antibody labelling methods:


1. Immunoenzyme method: An enzyme is used as the label.
Peroxidase, alkalin phosfatase, glucose oxidase
Chromogen (staining chemical) must be used
2. Immunofluorescence method: A fluorochrome is used as
the label.
AMCA, Fluorescein, FITC, Rodomine, TRITC, Texas Red.
Fluorescence microscope with appropriate filter or confocal
laser scanning microscope must be used to visualize.
3. Immunogold method: colloidal gold particles are used as
the label.
Usually used in electron microscopy.

Immunolabelling methods:
1. Direct method: The antigen directly binds to its specific
labelled antibody (primary antibody)
Fast to get the results
Labeling intensity is low
Used for kidney or skin biopsies.

Direct method

Immunolabelling methods:
2. Indirect method: Primary antibody is unlabelled. A
secondary antibody (which is labeled) is used.
Secondary antibody must be raised against the immunoglobulin of the
species which the primary antibody is made in.

Getting results takes longer


More sensitive
More economic

Indirect method

Fluorescence Method

Texas Red,
Rodamin,
Cy3

FITC,
Cy2

AMCA

Immunolabelling methods:
3. Unlabelled antibody methods:
Enzym-antienzym method
Peroxidase antiperoxidase (PAP)
Alkaline phosphatase - anti-Alkaline phosphatase (APAAP)
Most sensitive results
Widely used
Applied on paraffin, cryostat sections or on smears.

Peroxidase
antiperoxidase
(PAP)

Alkaline phosphatase anti-Alkaline


phosphatase (APAAP)

Immunolabelling methods:
5. Avidin-Biotin method:
Uses the high affinity of Avidin (glycoprotein) for biotin
(vitamin).
A complex of avidin-biotin-enzym (peroksidaz) is
necessary.
Streptoavidin can be used instead of avidin.
The secondary antibody is labelled with biotin which
inturn binds to avidin in the avidin-biotin-enzym complex.
Very high sensitivity
Used in research more than routine studies. It is longer and
more expensive.

In order to visualize the enzymes labelling the antibodies


with light microscope, enzyme substrate reactions, which
convert colorless chromogens into visible colored endproducts, is used.:
Peroxidase- hydrogen peroxide- diaminobenzidine
(DAB): BROWN
3-amino-9-ethylcarbazole (AEC): RED
4-chloro-1-naphthol (CN): KOYU
DARK MAV
BLUE

Multiple immunolabelling

Detecting more than one antigen in the same cell or on the same
tissue.
A combination of different single labelling methods is used.
Cross-reaction must be avoided:
Using primary antibodies raised in different species.
(not necessary if antigens of interest are localized in different
compartments of the cell such as cytoplasm vs nucleus.)
The secondary antibodies must be raised in the same species
such as donkey.
Specificity of the primary antibodies must be controled before.

Light Microscobe (two antigens, sometimes three)


Fluorescence (two or three) or confokal microscobe (two-five)
Combining two techniques (two-six)
Electron microscobe (usually two)

Radioisotope labels

Advantages
Flexibility
Sensitivity
Size

Disadvantages
Toxicity
Shelf life
Disposal costs

Enzyme labels

Advantages
Diversity
Amplification
Versatility

Disadvantages
Lability
Size
Heterogeneity

Fluorescent labels

Advantages
Size
Specificity
Sensitivity

Disadvantages
Hardware
Limited selection
Background

IgG

IgG

Confocal

Application in renal diseases


IgG

Immunohistochemistry

Aulanniam
University of Brawijaya

Where Immunohistochemistry is Used?

to detect:
Proteins, carbohidrates, nucleic acids, lipits
Types of the secreting cells
Membrane antigens
Structural antigens within the cytoplasm
Antigen localised in the nucleus

Immunohistochemistry is frequently used both in


experimental and diagnostical studies
Light, flouresans, confocal laser scanning or electron
microscope is used in order to visualize the labeled
antibody-antigen complex.

The aim of the immunohistochemistry

to perform most specific immunohistochemical


staining;
by cousing least damage on the cell or tissue,
by using least amount of antibody,
in shortest time,
with least backgroung staining.

Immunohistochemistry protocol -1

Before incubation with antibody:


Deparaffinization.
Removing the fixative: washing in buffer solutions
(phosphate buffer, Tris-HCl buffer, HEPES buffer, etc)
Neutralization of the endogeneus peroxidase
Blocking: covering the non-immunological sticky
sites on tissues (bovine serum albumine, nonimmune normal serum, gelatine, milk)
Blocking the surface tension: (Tween 20, Triton X-100,
NaCl)

Tissue Preparation

Fixation: Immersion or perfusion fixation


Neutral formaline
Paraformaldehyde
Paraformaldehyde ve picric acid (Bouins solution)
Sectioning:
Microtome: paraffin blocks
Cryostat: frozen tissue
Vibratome: fixed hard tissue
Sections on a slide (PAP Pen)
Floating sections

Immunohistochemistry protocol -2

Incubation:
Primary antibody: Used in a solution at different dilutions with
the blocking agent. The incubation time changes according to
the properties of the antigen or antibody as well as depending
on the temperature.
Secondary antibody: Must be raised in the species other
than the species of which the cells or tissues are taken or
the primary antibody is raised. It should specifically
recognize the immunoglobuline of the species in which the
primary antibody is raised.

Labelling: Immunoenzyme, immunoflourescence, immunogold


Microscopical analyses

Determining the Secretory


Contents of the
Neuroendocrine Neurones

The use of the Immunohistochemistry:

Intercellular antigens, for instance: immunoglobulines of


the kidney glomerular basal membrane
Cell surface antigens, tissue antigen for diagnosing
autoimmune diseases
Protein hormones in histopathological diagnosis
Soluble antigens of the cell
Diagnosis of the endocrine tumors
Small amounts of peptides in endocrine or neuroendocrine
cells
Immunodeposits
Tumoral markers
Tumor typing

The basic principle of


immunofluorescence
To use a fluorescent compound (usually
fluorescein) to detect the binding of antigen and
antibody
The Ab is labelled with the fluorescent compound
Under a fluorescence microscope, fluorescein
appears bright green wherever the binding occurs

Using the fluorescence microscope


Select the correct filter block for the
fluorescent compound
Fluorescence fades quickly under UV light;
try to limit the time of exposure to UV as
much as possible
Use high speed films for photography

Direct Immunofluorescence
The aim is to identify the presence and
location of an antigen by the use of a
fluorescent labelled specific antibody

Medical applications of direct IF


Renal diseases for evidence of immune
deposition
Skin diseases for evidence of immune
deposition
Detection of specific antigens, especially
those of infective organisms

Application in renal diseases


IgG

A section of kidney is placed on a slide; a


fluorescein-labeled antiglobulin (specific for IgG,
in this case) is added, then rinsed away
The presence of fluorescence in the glomeruli
indicates that IgG was deposited prior to the biopsy
IgG is deposited in granular clumps along the
capillary walls, enabling a diagnosis of
membranous glomerulonephritis in this case

Chemiluminescent labels

Advantages
Size
Sensitivity

Disadvantages
Hardware

Double labelling

Indirect Immunofluorescence

Indirect Immunofluorescence
The aim is to identify the presence of
antigen specific antibodies in serum. The
method is also be used to compare
concentration of the antibodies in sera.

Indirect Immunofluorescence
A known antigen is placed on a slide; the
patient's serum is added, then rinsed away.
A fluorescein-labeled antiglobulin is added,
then rinsed away.
The presence of fluorescence over the
antigen indicates the presence of antibodies
to this antigen in the patient.

Diagnosis of Bacterial Diseases

Clostridial diseases (direct)


Brucella canis (indirect)
Afipia catei, cat scratch disease (indirect)
Borrelia burgdorferi (indirect)
Coxiella burnetii, Q Fever (indirect)
Rickettsia rickettsiae, Rocky Mountain
Spotted Fever (indirect)

Diagnosis of Viral Diseases


rabies virus (direct)
bovine immunodeficiency-like virus (indirect)
canine coronavirus (indirect)
canine distemper (indirect)
feline infectious peritonitis (corona-) virus (direct)
porcine respiratory and reproductive syndrome
(indirect)

Diagnosis of Protozoal Diseases

Babesia species (indirect)


Ehrlichia species (indirect)
Toxoplasma gondii (indirect)
Trypanosoma cruzi (indirect)
Cryptosporidia/Giardia (direct)
Encephalitozoon cuniculi (indirect)
Neosporum caninum (direct, indirect)

Some examples
Indirect Immunofluorescence

Indirect Fluorescent Antibody Test for


Antibodies to Toxoplasma gondii
Toxoplasma organisms are killed and placed on the slide;
the patients serum is added, then washed away.
A fluorescein-labeled antiglobulin is added, then washed
away.
The presence of the green fluorescence outlining the T.
gondii organisms indicates the presence of antibodies in
the patient's serum.

Immune-Mediated Disorders
antinuclear antibody (ANA) test (for
diagnosis of systemic lupus erythematosus)
Direct fluorescent antibody test for
deposition of Abs in tissues, e.g. kidney,
skin

Indirect Fluorescent Antibody Test


for Antinuclear Antibodies
Cells from a cultured cell line are placed on a
slide; the patient's serum is added, then rinsed
away.
A fluorescein-labeled antiglobulin is added, then
rinsed away.
The presence of fluorescence in the nucleus of
these cells indicates the presence of antibodies to
nuclear antigens in the patient.

THANKS

ELISA
The ELISA technique is used widely to
detect and quantitate organisms and/or their
products in foods, and synopses of some of
these applications are presented below. For
more details, the cited references should be
consulted.

Enzyme-linked immunosorbent assay

Specimen

Substrate

2nd antibody
E

S
E

P
E

Microtiter well

ELISA (variation 1)
Specimen

Labeled antigen
E
S
E

P
E

Microtiter well

ELISA (variation 2)
Labeled antibody
E

Specimen
E
E

E
E

Microtiter well