What Is ELISA?

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.

Types Of ELISA

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

• 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

• 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

• Competitive ELISA helps to detect antigen concentration in a sample.

• The microtitre wells are coated with the antigen.

• 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.

• More the concentration of antigen in the sample, lesser the free antibodies available to interact with the antigen, which is coated in the well.

• 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.

Advantages Of ELISA

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.