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.