ELISA (Enzyme Linked Immunosorbent Assay)

//ELISA (Enzyme Linked Immunosorbent Assay)

ELISA (Enzyme Linked Immunosorbent Assay)

ELISA was introduced by Peter Perlman and Eva Engvall at Stockholm University, Sweden in 1971. It is a common laboratory technique which is usually used to measure the concentration of antibodies or antigens. ELISA is used for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones.ELISA readers or microplate readers spectrophotometrically measures and quantitates the color differences in the 12 wells of the plate, they emit light at one wavelength and measure the amount of light absorbed and reflected by an object. ELISA plate readers can also be used to measure the amount of luminescence and fluorescence produced by chemical dyes when these are exposed to light

ELISAs are typically performed in 96-well polystyrene or polyvinyl plate and are coated with either inactivated antigen or antibody. Antibodies or antigens present in the sample are captured by corresponding antigen or antibody immobilized on to the solid surface. The plate is then washed with a buffered solution containing detergent to wash unbound material from the plate. To detect the bound antibodies or antigens, a secondary antibody that is attached to an enzyme such as horse-radish peroxidase, beta-galactosidase or alkaline phosphatase are added to each well. After an incubation period, the unbound secondary antibodies are washed off.When a suitable substrate is added such as ortho-phenyldiaminedihydrochloride (for peroxidase), para-nitrophenyl phosphate (for alkaline phosphatase), the enzyme reacts with it to produce a color. Such substrate is called chromogenic substrate. This color produced is measurable as a function or quantity of antigens or antibodies present in the given sample. The optical density of color is measured at 450nm.

Types of ELISA
On the basis of binding structure between the Antibody and Antigen, there are 3 types of ELISA
1. Indirect ELISA
2. Sandwich ELISA
3. Competitive

1. Indirect ELISA
This test is used to detect and measure the concentration of an antibody in a sample In Indirect ELISA microtiter well is coated with an antigen. A sample containing primary antibody is added to the microtiter well and allowed to react with the immobilized antigen. After the well is washed, the presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary antibody that binds to the primary antibody. Unbound secondary antibody is then washed and a specific substrate for the enzyme is added. The enzyme hydrolyzes the substrate to form colored products. The amount of colored end product is measured by spectrophotometric plate readers that can measure the absorbance of all the wells of 96-well plate.

2. Sandwich ELISA
Sandwich ELISA can be used to detect antibody. In this technique, an antibody is immobilized on the microtiter well. A sample containing antigen is added to the well and allowed to react with the coated antibody, forming an antigen-antibody complex. After the well is washed, a second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. After washing unbound secondary antibody, a substrate is added to the plate which is hydrolyzed by the enzyme to form colored products.

3. Competitive ELISA
This test is used to measure the concentration of an antigen in a sample.In this test, the antibody is first incubated in a solution containing antigen. The antigen-antibody mixture is then added to an antigen coated microtitre well. The more the antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. After the well is washed, enzyme-conjugated secondary antibody specific for the primary antibody is added to determine the amount of primary antibody bound to the well. The higher the concentration of an antigen in the sample, the lower is the absorbance.

Advantages

• High specificity,
• Suitable for complex samples, since the antigen does not require purification prior to measurement.
• Flexibility and sensitivity.
• ELISA plate readers measure more samples in a shorter period of time than a spectrophotometer which measures one to six samples at a time.
• A range of labeled secondary antibodies are available commercially.
• Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection.
• Cost savings
• Different visualization markers can be used with the same primary antibody.

Application of ELISA
1. Indirect ELISA is used to detect the presence of serum antibodies against HIV, the causative agent of AIDS. This method allows the detection of Serum antibodies to HIV within 6 weeks of infection.
2. Presence of antigen or antibody in a sample can be detected.
3. Used in food industry to detect potential food allergens.
4. Allow the tracking of disease e.g.bird flu, common, colds, cholera, STD etc.

By | 2018-02-27T09:13:14+00:00 February 20th, 2018|Molecular Biology|Comments Off on ELISA (Enzyme Linked Immunosorbent Assay)

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