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Enzyme Linked Immunosorbent Assay : A Biotechnology TechniqueBY: Nidhi Uppangala | Category: Healthcare | Submitted: 2010-06-08 21:42:31
Article Summary: "Enzyme linked Immunosorbent assay also known as ELISA is quantitative immunological assay used to monitor the antigen- antibody reaction by measuring enzymes. Elisa term was first used in 1971 by Engwall and Perlma..."
Enzyme linked Immunosorbent assay also known as ELISA is quantitative immunological assay used to monitor the antigen- antibody reaction by measuring enzymes. Elisa term was first used in 1971 by Engwall and Perlma. ELISA is being used to screen HIV, as it is very sensitive and accurate.
Why Known as Enzyme Linked Immunosorbent Assay .....
1. Antigen of interest is absorbed onto the plastic surface, therefore sorbent.
2. Antigen of interest is identified by specific antibody, therefore immunological
3. This specific antibody is recognised by secondary antigen, which is linked to the enzyme, therefore the word enzyme linked
4. Substrate reacts with enzyme to give colored product
Basic Principle of ELISA:
1. Antigen antibody complex is detected using enzyme
2. Antigen-antibody complex is detected by the colored product produced by the enzyme using colorless substrate (chromogen)
3. Antigen or antibody can be detected based on the type of ELISA
Basic Procedure of ELISA:
1. Sample is loaded into the microplate strips and incubated for around five minutes, then rinse.
2. Primary antibody is added to the microplate strips and incubated for 5 minutes and rinsed.
3. Secondary antibody linked with enzyme are added to all wells, incubated for five minutes and rinsed.
4. Substrate specific for the enzyme is added and incubated. Colored product is formed and then it is being measured.
Types of ELISA:
Competitive ELISA microwell is coated with antibody, sample antigen and labelled antigen are added simultaneously, so the name competitive. Bound antibody-antigen-enzyme complex is inversely related to the concentration of antigen in the sample. Conjugated enzyme reacts with the colorless substrate to produce colored products. Competitive ELISA used to determine the small molecule antigens like T3, T4, and progesterone.
In this type of ELISA antigen present in the sample binds with the antibody captured in the microwell. The secondary antibody linked with the enzyme conjugate with the antigen to form Antibody-Antigen-Antibody/Enzyme sandwich. Here colored product formed by the enzyme reaction is directly proportional to the antigen present in the sample. This type of assay is used to determine the antigens such as tumor markers, hormone and serum proteins.
Advantages of Enzyme linked Immunosorbent assay:
1. Reagents used in ELISA are relatively cheap and have got long shelf life
2. Enzyme linked Immunosorbent assay is very specific and sensitive
3. While labelling or disposing of waste no radiations are used
4. Easy and quick assay
5. Enzyme linked Immunosorbent assay can be used to detect a variety of infections.
Disadvantages of Enzyme linked Immunosorbent assay:
1. Measurement of enzyme activity is more complex than measuring radioisotope activity
2. Plasma constituents may affect the activity of enzymes
3. ELISA kits which are commercially available are not cheap
4. Enzyme linked Immunosorbent assay is very specific to a particular antigen therefore cannot be used to detect other antigens
5. False results are possible because of mutated or altered antigen
Applications of Enzyme linked Immunosorbent assay:
1. ELISA can be used to detect hormone, protein, infectious agents, drug markers, tumor markers, serum proteins.
2. ELISA can be used in new born screening
3. ELISA can be used in clinical research
4. ELISA can be used in vaccine quality control
5. Antibody detection
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