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What is the ELISA Assay Principle?

As we all know that enzyme-linked immunosorbent assay or ELISA is a procedure of catching the target antigen present in the samples with a particular antibody. It is also used for measuring the antigen by working with an enzyme reaction using its substrate given in the ELISA kit. In ELISA, many antigen-antibody mixtures are used.

It includes an enzyme-labeled antigen or antibody, and the activity of the enzyme is measured through colors. The enzyme activity is measured with a parasite that changes its color when evolved by the enzyme. The absorption of light takes place during this process. You can know more about the ELISA assay principle at


An overall ELISA principle consists of few steps. The first is to coat the microtiter plate wells with antigen. The second is to block any outside elements to avoid variation in results. The third is the addition of primary antibody into the molds present in the ELISA kit. The fourth step includes the addition of a secondary antibody conjugated to an enzyme.

The last step is to record the response of a substrate using the enzyme to make the color change. In direct ELISA, the targeted protein is trapped on the surface of the microplate and incubated with an enzyme-labeled antibody into the target protein. In indirect ELISA, the trapped target protein is incubated with an antibody to the target protein with a secondary antibody from the principal antibody.

Sandwich ELISA also contains trapped target protein and then this protein is linked with a different targeted protein-specific antibody, which can be labeled with the help of an enzyme. In competitive ELISA, the antibody specific for a target protein is trapped and incubated with samples containing the target protein. So, the ELISA can be used to discover the existence of antigens present in the given sample.