Download - Antibody based techniques
ANTIBODY BASED TECHNIQUES
PRIYANKA PATHANIA
BTECH. BIOTECH
What are antibodies...??
An antibody (Ab) , also known asimmunoglobulin (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.
Antigen and Antibody Reaction
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 containing an unknown quantity of that same antigen is added.
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 tranferred 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.