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ANTIBODY BASED
TECHNIQUES
PRESENTED BY :
PRIYANKA PATHANIA
BTECH. BIOTECH (5TH
SEM)
12btb030
What are antibodies...??
An antibody (Ab) , also known as
immunoglobulin (Ig), is a large “Y”
shaped protein produced by plasma
cells that is used by the immune
system to identify and neutralize
foreign objects like bacteria and viruses.
Each antibody recognizes a specific
antigen unique to its target.
Antibodies (Immunoglobulins)
Monoclonal antibodies :
(mAb) are antibodies that are identical
because they are produced by one type of
immune cells, all clones of a single parent cell.
Polyclonal antibodies :
Are the antibodies that are derived from
different cell lines. They differ in amino acid
sequence.
What is an antigen...??
An antigen (Ag), is a substance or any
molecule that induces an immune response in
the body.
An antigen is often foreign or toxic to the body
for example bacteria or virus, which once in
the body, attracts and is bound to a respective
and specific antibody.
INTRODUCTION
The antigens and the antibodies combine
specifically with each other. This interaction
between them is called Antigen-Antibody reaction.
It may be abbreviated as Ag – Ab reaction.
These reactions form the basis for detection of
infectious disease causing agents and also some
non-specific Ag’s like enzymes.
Types of Ag- Ab reactions...
RIA – Radioimmuno Assay
ELISA – Enzyme Linked Immuno Sorbent
Assay
Western blots
Precipitation Reactions
Radioimmunoassay (RIA)
Involves the separation of a protein (from a
mixture) using the specificity of antibody -
antigen binding and quantify it using
radioactivity
The technique was introduced in 1960 by Berson
and Yalow as an assay for the concentration of
insulin in plasma.
INTRODUCTION
Radioimmunoassay (RIA) is a very
sensitive in vitro assay technique used to
measure concentrations of antigens (for
example, hormone levels in the blood) by use
of antibodies.
The RAST test (radio allergosorbent test) is an
example of radioimmunoassay. It is used to
detect the causative allergen for an allergy
To perform a radioimmunoassay, a known
quantity of an antigen is made radioactive,
frequently by labeling it with gamma-
radioactive isotopes of iodine attached to
tyrosine.
This radiolabeled antigen is then mixed with a
known amount of antibody for that antigen, and
as a result, the two specifically bind to one
another.
Then, a sample of serum from a patient
This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody binding sites. As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter.
Radioimmunoassay (RIA)
Advantages & Disadvantages of
RIA
Advantages
Highly specific: Immune reactions are specific
High sensitivity : Immune reactions are sensitive
Possible to detect picograms of Ag
Sepharose beads used in RIA are reuseable
Disadvantages
Radiation hazards: Uses radio labelled reagents
Requires specially trained persons
Labs require special license to handle radioactive material
Requires special arrangements for storage of radioactive material radioactive waste disposal.
ENZYME LINKED
IMMUNOSORBENT ASSAY
(ELISA)
INTRODUCTION TO ELISA
ELISA, or enzyme-linked immunosorbent assay, are quantitative immunological procedures in which the Ag- Ab reaction is monitored by enzyme measurements.
The term ELISA was first used by Engvall & Perlma in 1971.
The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.
Elisa Plate
Microtitre wells
Generally, 96 wells
Marked on one side
alphabetically
Numerically on other
side
Comes with the kit
BASIC PRINCIPLE OF ELISA
Use an enzyme to detect the binding of antigen (Ag) antibody (Ab).
The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag : Ab binding.
An ELISA can be used to detect either the presence of Antigens or antibodies in a sample depending how the test is designed.
ELISA was dveloped in 1970 and became rapidly accepted
TYPES OF ELISA
• Indirect elisa
• Sandwich elisa
• Competetive elisa
Comparison between Indirect, Sandwich & Competitive
ELISA
Equipments for performing the
ELISA test
ELISA READER
Advantages of ELISA
Reagents are relatively cheap & have a long shelf life
ELISA is highly specific and sensitive
No radiation hazards occur during labelling or disposal of waste.
Easy to perform and quick procedures
Equipment can be inexpensive and widely available.
ELISA can be used to a variety of infections.
Disadvantages of ELISA
Measurement of enzyme activity can be more complex than measurement of activity of some type of radioisotopes.
Enzyme activity may be affected by plasma constituents.
Kits are commercially available, but not cheap
Very specific to a particular antigen. Won’t recognize any other antigen
False positives/negatives possible, especially with mutated/altered antigen
APPLICATIONS OF ELISA
detection of Mycobacterium antibodies in
tuberculosis
detection of hepatitis B markers in serum
detection of HIV antibodies in blood samples
It has also found applications in
the food industry in detecting potential food
allergens, such
as milk, peanuts, walnuts, almonds, and eggs.
Western blot (Immunoblotting)
Blots are techniques for transferring DNA ,
RNA and proteins onto a carrier so they can be
separated, and often follows the use of a gel
electrophoresis. The Southern blot is used for
transferring DNA, the Northern blot for RNA
and the western blot for PROTEIN.
A technique for detecting specific proteins
separated by electrophoresis by use of labeled
antibodies.
Procedure
In Western blotting, a protein mixture is
separated on a polyacrylamide slab gel
that has been treated with sodium dodecyl
sulfate, a dissociating agent.
The protein bands are then transferred to
a nitrocellulose membrane by capillary
blotting.
And the individual protein bands are
identified by flooding the blot with antigen-
specific
primary antibody, followed by incubation and
washing and finally addition of radiolabelled or
enzyme labelled secondary antibody specific
for the primary antibody.
The antigen-antibody complexes (bands) that
form are visualised by autoradiography.
Western Blotting Procedure
Applications
The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody.
Western blot can also be used as a confirmatory test for Hepatitis B infection.