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Monoclonal
Various classes of
antibodies are present
(IgG,IgM,and so on)
Reproducibility and
standardization difficult
Highly reproducible
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.
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)
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.
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.
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
Immunohistochemistry
Aulanniam
University of Brawijaya
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 protocol -1
Tissue Preparation
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.
Direct Immunofluorescence
The aim is to identify the presence and
location of an antigen by the use of a
fluorescent labelled specific antibody
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.
Some examples
Indirect Immunofluorescence
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
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.
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