ELISA is the basic assay technique, known as enzyme-linked immunosorbent assay (also referred to as EIA: Enzyme Immunoassay) that is carried out to detect and measure antibodies, hormones, peptides and proteins in the blood.
Antibodies are blood proteins produced in response to a specific antigen. It helps to examine the presence of antibodies in the body, in case of certain infectious diseases.
ELISA is a distinguished analysis compared to other antibody-assays as it yields quantitative results and separation of non-specific and specific interactions that take place through serial binding to solid surfaces, which is normally a polystyrene multiwell plate.
ELISA tests can be classified into three types depending upon the different methods used for binding between antigen and antibodies, namely:
Indirect ELISA – Antigen is coated to the microtiter well
Sandwich ELISA – Antibody is coated on the microtiter well
Competitive ELISA – Microtiter well which is antigen-coated is filled with the antigen-antibody mixture.
Indirect ELISA detects the presence of an antibody in a sample.
The antigen is attached to the wells of the microtitre plate.
A sample containing the antibodies is added to the antigen-coated wells for binding with the antigen.
The free primary antibodies are washed away and the antigen-antibody complex is detected by adding a secondary antibody conjugated with an enzyme that can bind with the primary antibody.
All the free secondary antibodies are washed away. A specific substrate is added which gives a coloured product.
The absorbance of the coloured product is measured by spectrophotometry.
Sandwich ELISA helps to detect the presence of antigen in a sample.
The microtitre well is coated by the antibody.
The sample containing the antigen is added to the well and washed to remove free antigens.
Then an enzyme-linked secondary antibody, which binds to another epitope on the antigen is added. The well is washed to remove any free secondary antibodies.
The enzyme-specific substrate is added to the plate to form a coloured product, which can be measured.
Competitive ELISA helps to detect antigen concentration in a sample.
Antibodies are incubated in a solution having the antigen.
The solution of the antigen-antibody complex is added to the microtitre wells. The well is then washed to remove any unbound antibodies.
The enzyme-linked secondary antibody is added to detect the number of primary antibodies present in the well.
The concentration is then determined by spectrophotometry.
Following are some of the advantages of the ELISA technique:
Results fetched from ELISA gives an accurate diagnosis of a particular disease since two antibodies are used.
Can be carried out for complex samples as the antigen is not required to get purified to detect.
It is highly responsive since direct and indirect analysis methods can be carried out.
It is a rapid test, yields results quickly.
Possible detection for ELISA ranges from the quantitative, semi-quantitative, standard curve, qualitative, calibration curve models etc.
Easier to perform and uncomplicated process as compared to other assays which require the presence of radioactive materials.
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