Labelling_Conjugated

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Labelling_Conjugated
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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
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• Antibody labelling methods
• Immunoenzyme method An enzyme is used as the
  label.
• Peroxidase, alkalin phosfatase, glucose oxidase
• Chromogen (staining chemical) must be used
• 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.

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• Immunolabelling methods
• 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.

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Direct method
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• Immunolabelling methods
• 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

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Indirect method
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Fluorescence Method
Texas Red, Rodamin, Cy3
FITC, Cy2
AMCA
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• Immunolabelling methods
• Protein A method
• 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.

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Peroxidase antiperoxidase (PAP)
Alkaline phosphatase - anti-Alkaline phosphatase
(APAAP)
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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.
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• 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 MAVI

BROWN
RED
DARK BLUE
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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)

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Radioisotope labels

• Advantages
• Flexibility
• Sensitivity
• Size

• Disadvantages
• Toxicity
• Shelf life
• Disposal costs

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Enzyme labels

• Advantages
• Diversity
• Amplification
• Versatility

• Disadvantages
• Lability
• Size
• Heterogeneity

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Fluorescent labels

• Advantages
• Size
• Specificity
• Sensitivity

• Disadvantages
• Hardware
• Limited selection
• Background

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IgG
IgG
Confocal
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Application in renal diseases
IgG
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Immunohistochemistry
Aulanniam University of Brawijaya
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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.

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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.

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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)

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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

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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

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Determining the Secretory Contents of the
Neuroendocrine Neurones
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• 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

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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

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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

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Direct Immunofluorescence

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

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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

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Application in renal diseases
IgG
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• 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

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Chemiluminescent labels

• Advantages
• Size
• Sensitivity

• Disadvantages
• Hardware

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Double labelling
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Indirect Immunofluorescence
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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.

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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.

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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)

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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)

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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)

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Some examples

• Indirect Immunofluorescence

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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.

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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

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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.

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THANKS
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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.

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Enzyme-linked immunosorbent assay
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ELISA (variation 1)
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ELISA (variation 2)