ELISA

Enzyme Linked Immunosorbent Assay

Purpose of ELISA

To detect antibodies in your blood or urine.

To see if you have been exposed to a disease

How Your Body Works

The key to a healthy immune system is its remarkable ability to distinguish between the body’s own cells (self) and foreign cells (nonself).

How Your Body Works

You are exposed to a foreign substance (something you don’t have in your body);

Ex: bacteria, viruses, fungi, protein, etc. These are called antigens.

Anything that can trigger this immune response is called an antigen.

Cont.

Your body reacts (immune response) by producing antibodies.

Antibodies are proteins.

Antibdies Within days of exposure, your body

produces millions of antibodies. Each antibody is made up of two identical heavy chains and two identical light chains, shaped to form a Y. It is these unique contours in the antigen-binding site that allow the antibody to recognize a matching antigen.

Antibodies (variable region) recognize antigens.

Antibodies attach themselves to their target antigens.

Antibodies ‘flag’ the invaders (antigens)

HIV Protein

X-ray diffraction analysis of a human immunodeficiency virus (HIV-1) capsid (CA) protein shows that each monomer within the dimer consists of seven alpha-helices, five of which are arranged in a coiled coil-like structure.

Antibody

The structure of an intact monoclonal antibody for phenobarbital, subclass IgG1, has been determined to 3.2 A resolution by X-ray crystallography. .

How are Antibodies Made

Use of animals!!!!!!!!! Rabbits, mice, sheep, chickens,

goats 1. Animals are injected with

antigen>> 2. Body will produce antibodies>>

3. Antibodies float in blood. 4. Blood is removed from from the

animal. 5. antibodies are purified from

blood.

Primary vs. Secondary

If the antibodies are used to confirm a disease (with ELISA or another other immunoassay), the antibodies are called primary antibodies.

Secondary antibodies recognize and bind to primary antibodies, which are antibodies from another species.

Secondary Antibodies (ab)

Prepared by injecting antibodies made in one species into another species.

Secondary ab are different enough from primary ab that they will be recognized as foreign proteins

Immune response is produced in second species.

Example Human antibody injected into

rabbit. Rabbit produces antibodies

that recognize and bind to human antibodies.

ELISA assay -one example

The second antibodies are conjugated (linked) to enzyme HRP, horseradish peroxidase.

This conjugate produces blue color in the presence of substrate TMB (tetramethyl benzidine)

Immune Response

Exposure to disease immune system produced antibodies

first antibody is called Immunoglobulin M (IgM). Can be detected in blood.

Primary ans Secondary exposure

Eva Engvall is one of the scientists who invented ELISA in 1971.

Dr. Engvall earned her Ph.D. from the University of Stockholm in 1975.

Her postdoctoral work was done at the University of Helsinki and City of Hope National Medical Center in California, where she was subsequently appointed to staff. Dr. Engvall was recruited to The Burnham Institute for Medical Research in 1979. For 1993-1996, Dr. Engvall held joint appointments at Burnham Institute for Medical Research and as Chairperson of the Department of Developmental Biology at Stockholm University. Dr. Engvall received an honorary degree in Medicine from the University of Copenhagen in November 1994.