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Labelling_Conjugated

Lecture in Faculty of Veterinary Medicine University of Brawijaya

by :

Dyah Kinasih Wuragil, S.Si, MP, M.Sc

VETERINARY MEDICINE SCHOOL

UNIVERSITY OF BRAWIJAYA

MALANG

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.

Sekondary 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

AMCA

FITC,

Cy2

Immunolabelling methods:

3. Protein A method:

4. 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 end-

products, is used.:

• Peroxidase- hydrogen peroxide- diaminobenzidine

(DAB):

• 3-amino-9-ethylcarbazole (AEC):

• 4-chloro-1-naphthol (CN): KOYU MAVİ

BROWN

RED

DARK 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

Aulanni’am

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, non-

immune 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 (Bouin’s solution)

• Sectioning:

• Microtome: paraffin blocks

• Cryostat: frozen tissue

• Vibratome: fixed hard tissue

• Sections on a slide (PAP Pen)

• Floating sections

• 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

Immunohistochemistry protocol -2

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 patient’s 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

Microtiter well

E E E E E

Specimen 2nd antibody E

Substrate

S P

ELISA (variation 1)

Microtiter well

Specimen Labeled antigen

E

E E E

S P

ELISA (variation 2)

Microtiter well

Specimen Labeled antibody E

E E E E

E E E