Enzyme Linked Immuno Sorbent Assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA), is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample.

The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.

Enzyme Linked Immunosorbent Assays (ELISA) are those that are based on the measurement of an enzymatic reaction associated with immune complexes. In any particular assay, the enzyme may be linked to either the antigen or the antibody

Principle

Antibody specific for a protein of interest is attached to a polymeric support such as a sheet of PVC

Procedure

Formation of the antibody-antigen complex

Formation of 2nd antibody-antigen complex

+Add sample (A drop of cell extract or a sample of serum is laid on the sheet)

Wash to remove unbound molecules

+Add 2nd antibody(which is specific for a different site on the antigen)

+Add enzyme that can be detected with high sensitivity

Wash to remove unbound 2nd antibody

The amount of protein in sample α to the amount of 2nd antibody bound with antigen

Advantages of enzyme addition

The sensitivity of the assay can be enhanced if the second antibody is attached to an enzyme such as alkaline phosphatase

This enzyme can rapidly convert many molecules of an added colorless substrate into colored products.

Enzyme helps to detected antigen-antibody complex with high sensitivity

Attachment of 1st antibody (Ab1) into a PVC sheet

Addition of sample (target protein, ) Wash to remove unbound molecules

Polymer Support

Addition of 2nd antibody (AB2)with enzyme, )

Wash to remove unbound 2nd Ab

Polymer Support

Ab1

Ab2

Polymer Support

Enzyme

Substrate for that enzyme,

Advantages High sensitivity

Greater reproducibility

Requirement of minimal reagents

Qualitative (e.g., HIV testine) and quantitative ( e.g., therapeutic drug monitoring) assay can be done

Well can be coated with antigens or antibody

High speed

No radiation hazards

Applications1. Analysis of hormones, vitamins, metabolites and

diagnostic markers e.g., ACTH FSH Tri-iodothyronine (T3 ) Thyroxin (T4 ) Glucagon Insulin Testosterone Vitamin B12

Prostaglandins Glucocorticoids

2. Therapeutic drug monitoring e.g., Barbiturates Digoxin Digitoxin Morphine

3. Diagnostic procedures for detecting infection e.g., HIV Hepatitis A & B

Although the RIA technique is extremely sensitive and extremely specific, it requires specialized equipment, well trained manpower, special precautions and licensing, since radioactive substances are used.

In the case of ELISA method, where the antigen-antibody reaction is measured using colorimetric signals instead of a radioactive signal.

In ELISA method, there is no radiation hazards

Advantages of ELISA over RIA

However, because of its robustness, consistent results and low price per test , RIA methods are convenient than Elisa

Advantages of RIA over ELISA