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Enzyme Linked Immunosorbent Assay (ELISA): Procedure, Applications, TypesBY: Amna Adnan | Category: Biotechnology-products | Submitted: 2010-06-21 21:33:53
Enzyme linked Immunosorbent assay is a biological technique which is used in the detection and measurement of the antibody and antigen in the body. This technique is commonly used to treat the disease of cancer or autoimmune diseases and is also useful in plant pathology. The biological samples which are used during this test are usually blood, urine or some cell extracts. The basic procedure of this technique is to use an antibody, which finds an antigen corresponding to this antibody and binds to it. This bond of antibody-antigen is recognized by another antibody. This antibody is joined to an enzyme which is responsible for catalyzing the mixture of the reaction.
How ELISA is performed?
Following steps are involved in the ELISA test:
1) An antibody is taken which has the property of binding to a particular antigen.
2) A polystyrene microtiter plate is usually used to keep the antigen immobilized. This plate is made of solid material and is fixed.
3) When the antigen is immobilized by using a solid surface, then an antibody is added in the reaction. This antibody has the ability to detect the antigen.
4) When the antibody enters the reaction, it finds the antibody, attaches to it and forms a complex.
5) There are two methods of finding the detection antibody. Either an enzyme is used to link to the antibody or another antibody that is secondary antibody is used to detect the detection antibody. The secondary antibody is also linked to an enzyme through bioconjugation.
6) During the whole process, the plate is washed regularly with a mild detergent, so that the proteins or those antibodies can be removed which are not needed for the process.
7) When the plate is washed for the last time, then an enzymatic substrate is added in the process, so that the quantity of the antigen in the sample can be indicated. This enzyme releases a signal through which detection of quantity of antigen is made possible.
Applications of ELISA:-
In biotechnology processes, ELISA has significant value. It can be used to detect antigens or antibodies in the samples. It is also useful in determining the concentrations of serum antibody during the test of HIV or West Nile Virus. Food industry has also taken advantage of this field as it is used to detect the allergens in the foods like milk, peanut, eggs, almonds and walnuts and almost all the foods which are rich with proteins. Certain drugs can be analyzed by using the method of ELISA in the field of toxicology.
Types of ELISA:-
Competitive ELISA is involved in the quantification of antigens. In this method, through incubation, free antigens and antibodies are brought together, they form a complex. This complex is brought near the antigen coated surface in the plate. Then a secondary antibody is added which detects this complex. Through a specific signal by using an enzyme, this complex can be detected. The unnecessary antigens and antibodies are washed out and the complex is further processed.
In this process, the detection of antigen takes place between two plates of antibodies that is why it is called as sandwich ELISA. There should be two binding sites of the antigen so that both the antibodies can attach to the antigen. This type of ELISA is used to quantify the proteins or polysaccharides.
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