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.