1. 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.

2. 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

3. 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 2 nd 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 2 nd antibody(which is specific for a different site on the antigen) +Add enzyme that can be detected with high sensitivity Wash to remove unbound 2 nd antibody The amount of protein in sample α to the amount of 2 nd antibody bound with antigen

4. 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

5. Attachment of 1 st antibody (Ab1) into a PVC sheet Addition of sample (target protein, ) Wash to remove unbound molecules Polymer Support Addition of 2 nd antibody (AB2) with enzyme, ) Wash to remove unbound 2 nd Ab Polymer Support Ab1 Ab2 Polymer Support Enzyme Substrate for that enzyme,

6. 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

7. Applications 1.Analysis of hormones, vitamins, metabolites and diagnostic markers e.g.,  ACTH  FSH  Tri-iodothyronine (T 3 )  Thyroxin (T 4 )  Glucagon  Insulin  Testosterone  Vitamin B 12  Prostaglandins  Glucocorticoids 2.Therapeutic drug monitoring e.g.,  Barbiturates  Digoxin  Digitoxin  Morphine 3.Diagnostic procedures for detecting infection e.g.,  HIV  Hepatitis A & B

8.  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. sensitivespecific  In the case of ELISA method, where the antigen- antibody reaction is measured using colorimetric signals instead of a radioactive signal. In the case of ELISAcolorimetric  In ELISA method, there is no radiation hazards Advantages of ELISA over RIA

9. However, because of its robustness, consistent results and low price per test, RIA methods are convenient than Elisa Advantages of RIA over ELISA