ELISAELISA

BY : VIVEKVIVEK MSc BiotechMSc Biotech..

Enzyme Linked Enzyme Linked Immunosorbent Immunosorbent

AssayAssay

Technique used to detect (assay) specific molecules (e.g. proteins & carbohydrates) in samples.

Immunological technique: uses antibodiesantibodies..

Quantitative.

Very sensitive.

Commonly used in medicine and scientific research.

Recognise and bind to molecules (antigens) on foreign particles, marking them for destruction by T-lymphocytes.

Each antigen may generate several antibodies for different sites (epitopes) on antigen.

ANTIBODIESANTIBODIES Proteins secreted by B-lymphocytes (type of white blood cell), in vertebrates.

ANTIGENANTIGEN Is present naturally in the body like

hormones.

Is manufactured in special disease status for example human chorionic gonadotrophin hormone (HCG) which is normally produced by cells of the placenta in pregnancy is found in the body in some types of cancer.

Is not present in the body in normal condition like drugs.

AntigensAntigensA substance that when introduced into the

body stimulates the production of an antibody.

ImmunoassayImmunoassayA laboratory technique that makes use of the

binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample.

AnalyteAnalyteThe sample being analyzed and in

immunoassays the analyte is either Antibody or Antigen

1 .Antigen of interest is absorbed on to plastic surface (‘sorbent’).

2. Antigen is recognised by specific antibody (‘immuno’).3. This antibody is recognised by second antibody

(‘immuno’) which has enzyme attached (‘enzyme-linked’).4. Substrate reacts with enzyme to produce product,

usually coloured .

Coloured product = measure (assay) of antigen present

Peroxidase from horseradishAlkaline phosphatase from E. coli

-galactosidase from E. coli

React with a colourless substrate to produce a coloured product.

Must work fast at room temperature so the colour develops quickly.

Substrate

Primary antibody

Enzyme

Secondary antibody

Different antigens in sample

Coloured product

ELISA TECHNIQUEELISA TECHNIQUE Is a biochemical technique used mainly

in immunology to detect the presence of an antibody or an antigen in a sample.

The technique is divided into

1- Competitive ELISA 2- Sandwich ELISA (also called direct

ELISA)3- Indirect ELISA

COMPETITIVE ELISACOMPETITIVE ELISA

The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal.

SANDWICH ELISASANDWICH ELISA The ELISA plate is coated with

Antibody to detect specific antigen

SANDWICH ELISASANDWICH ELISA Prepare a surface to which a known

quantity of capture antibody is bound.

Block any non specific binding sites on the surface

Apply the antigen-containing sample to the plate.

SANDWICH ELISA-CONTSANDWICH ELISA-CONT Wash the plate, so that unbound antigen is

removed.

Apply enzyme linked primary antibodies as detection antibodies which also bind specifically to the antigen.

Wash the plate, so that the unbound antibody-enzyme conjugates are removed.

Apply a chemical which is converted by the enzyme into a coloured product.

Measure the absorbance of the plate wells to

determine the presence and quantity of antigen.

SANDWICH ELISASANDWICH ELISA

INDIRECT ELISAINDIRECT ELISA The protein antigen to be tested for

is added to each well of ELISA plate, where it is given time to adhere to the plastic through charge interactions.

A solution of non-reacting protein is added to block any plastic surface in the well that remains uncoated by the protein antigen.

INDIRECT ELISA-CONTINDIRECT ELISA-CONT Then the serum is added, which contains a

mixture of the serum antibodies, of unknown concentration, some of which may bind specifically to the test antigen that is coating the well.

Afterwards, a secondary antibody is added, which will bind to the antibody bound to the test antigen in the well. This secondary antibody often has an enzyme attached to it

A substrate for this enzyme is then added. Often, this substrate changes colour upon reaction with the enzyme. The colour change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen.

The higher the concentration of the primary antibody that was present in the serum, the stronger the colour change. Often a spectrometer is used to give quantitative values for colour strength.

1 .Add antigen7 .Add substrate

for enzyme

2 .Wash with water

4 .Wash with water

3 .Add primary antibody

6 .Wash with water

5 .Add secondary antibody

8 .Observe colour

development

Uses of ELISAUses of ELISA Disease detection in people, animals and

plants (e.g. HIV in humans) .

Detection of allergens in food, e.g. peanuts.

Detection of illegal drugs in humans.

Detection of hormones, e.g. pregnancy testing kits.

ELISA in ELISA in BiotechnologyBiotechnology Higher Biology, Biotechnology and Human BiologyE.g. Biotechnology: production & production & use of monoclonal antibodiesuse of monoclonal antibodies

Advanced Higher Biology :Biotechnology UnitEnvironmental Biology UnitInvestigations

Monoclonal antibody productionMonoclonal antibody production )hybridoma technologyhybridoma technology(

Fuse B-lymphocytes

with myeloma cells

Inject mouse with

antigen

Grow mouse myeloma (tumour)

cells in culture

Obtain Mouse spleen

B-lymphocytes

Antibody-producing hybridoma cells