Enzyme-Linked Immunosorbent Assay (ELISA) is a biochemical assay technique used primarily in immunology. It is a plate-based assay designed for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones
ELISA can be used to detect the presence of antigens that are acknowledged by an antibody or It will be used to test for antibodies that recognize an antigen.
A general Elisa Test Procedure is a five-step process involves coat the microtiter plate wells with antigen; block all unbound sites to prevent false positive results; add primary antibody (e.g. rabbit monoclonal antibody) to the wells; add secondary antibody conjugated to an enzyme (e.g. anti-mouse IgG); finally reaction of a substrate with the enzyme to produce a colored product, thus indicating a positive reaction.
Applications of ELISA
- Serum Antibody Concentrations
- Detecting potential food allergens (milk, peanuts, walnuts, almonds, and eggs)
- Tracking the spread of disease e.g. HIV, bird flu, common, colds, cholera, STD, etc
- Detections of antigens e.g. pregnancy hormones, drug allergen, GMO, mad cow disease
- Detection of antibodies in a blood sample for past exposure to disease e.g. Lyme Disease, trichinosis, HIV, bird flu
Types of ELISA Tests
There are five types of ELISA methods which include Direct ELISA, Indirect ELISA, Competitive ELISA, Multiplex ELISA, and Sandwich ELISA
In the sandwich ELISA test, the antigen is taken as a substance and is covered with two layers of antibody, i.e., capture antibody & detection antibody and then the result is calculated. There are two types of antibodies used in the Sandwich Elisa Protocol test - monoclonal and polyclonal. Examples of usage of Sandwich ELISA tests are Home pregnancy tests.
Advantages
- High specificity, since two antibodies are used the antigen is specifically captured and detected.
- Suitable for complex samples
- Flexibility and sensitivity, as both direct and indirect detection methods can be used
ELISA is a plate-based assay technique. Along with the enzyme-labeling of antigens or antibodies, the technique involves following three principles in combination which make it one of the most specific and sensitive than other immunoassays to detect the biological molecule:
- An immune reaction i.e. antigen-antibody reaction.
- Enzymatic chemical reaction i.e. enzyme catalyzes the formation of colored product from the colorless substrate.
- Signal detection and Quantification i.e. detection and measurement of color intensity of the colored products
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From the above discussion, we can conclude that the ELISA technique is used as a diagnostic tool in medicines or pathology related to plants. It has proved to be a good quality control check in industries involving food processing. Elisa test is conducted on the antigen and antibody concentrations. Whatever type of test is used, ELISA tries to give accurate results.
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ELISA principle and procedure is a technique for the target antigen (or counteracting agent) catch in tests utilizing a particular neutralizer (or antigen), and of target particle recognition/quantitation utilizing a catalyst response with its substrate.In ELISA, different antigen-counter acting agent mixes are utilized, continually including a chemical marked antigen or immunizer, and compound action is estimated colour metrically.The protein action is estimated utilizing a substrate that changes shading when adjusted by the chemical.
Light retention of the item framed after substrate expansion is estimated and changed over to numeric qualities.Contingent upon the antigen-counter acting agent mix, the test is known as an immediate ELISA protocol, backhanded ELISA protocol, sandwich ELISA protocol, aggressive ELISA protocol and so forth.Direct ELISAAn objective protein (or an objective neutralizer) is immobilized on the outside of microplate wells and brooded with a catalyst named immunizer to the objective protein (or a particular antigen to the objective counteracting agent).
In the wake of washing, the movement of the microplate well-bound catalyst is estimated.Indirect ELISAAn objective protein is immobilized on the outside of microplate wells and brooded with an immune response to the objective protein (the essential counteracting agent), trailed by an optional immunizer against the essential neutralizer.
Subsequent to washing, the movement of the microplate well-bound chemical is estimated.ELISA sample preparation requires a bigger number of steps than direct ELISA, named auxiliary antibodies are financially accessible, taking out the need to name the essential immunizer.Sandwich ELISA principleAn immunizer to an objective protein is immobilized on the outside of microplate wells and brooded first with the objective protein and after that with another objective protein-explicit neutralizer, which is named with a chemical.
Subsequent to washing, the action of the microplate well-bound chemical is estimated.
The immobilized counteracting agent (orange) and the compound marked immunizer (green) must perceive distinctive epitopes of the objective protein.Aggressive ELISAA counteracting agent explicit for an objective protein is immobilized on the outside of microplate wells and hatched with tests containing the objective protein and a known measure of catalyst marked target protein.After the response, the action of the microplate well-bound compound is estimated.At the point when the antigen level is high, the dimension of counteracting agent bound compound named antigen is lower and the colour is lighter.
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