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Difference Between Direct & Indirect ELISA

An enzyme-linked immunosorbent assay (ELISA) involves the reaction of an antibody-enzyme complex with an antigen or antibody held immobile on a solid surface. On incubation of the conjugated enzyme with a suitable substrate, a product is formed. The formation of a product helps to detect presence of the antigen or antibody and its quantity provides a measure of the amount of antigen-antibody reaction that occurred. The ELISA test may be performed in two forms: direct and indirect.

  1. Test Format

    • In the direct ELISA test, a primary antibody is held on the walls of a microtiter plate. When the sample suspected to contain the antigen is added, there is an antigen-antibody reaction. Next, an enzyme-linked secondary antibody that is capable of reacting with the antigen is added. If antigen is present in the sample, it binds to this enzyme-linked antibody. When a colorless substrate is added, if a color develops, it indicates the presence of antigen. In the indirect ELISA, antigen is held on the walls of the microtiter plate. When a sample suspected to contain antibodies is added, an antigen-antibody reaction occurs. Next, an enzyme-linked secondary antibody capable of reaction with the unbound region of the antibody is added. When the colorless substrate is added, it leads to development of color if the sample used contains antibodies.

    Ease of Testing

    • A wide range of labeled secondary antibodies are commercially available. Besides, it is possible to create several primary antibodies in a given species and use the same secondary antibody for detection. This makes the indirect ELISA easy to perform. In the direct ELISA, the primary antibodies have to be specifically prepared to react with the specific antigen suspected to be present in the sample. This makes the direct ELISA disadvantageous compared to the indirect test.

    Rapidity of Testing

    • The direct ELISA test is quick in comparison to the indirect test because it uses only a single antibody. The indirect ELISA requires an additional step of incubation with a second antibody during the test procedure, which causes a delay in obtaining results.

    Sensitivity

    • The direct ELISA test is less sensitive than the indirect form of the test because there is much less signal amplification. Besides, the labeling of the primary antibody with an enzyme may impair its immonoreactivity profile. In case of the indirect ELISA, the primary antibody is not labeled and therefore, it retains its immunoreactivity. Besides, each primary antibody has many epitopes that can bind with the secondary antibody; this permits amplification of the signal, improving the method's sensitivity. However, there is a chance of cross reactivity between the antigen and the secondary antibody, leading to an error in the results. There is no such possibility in the direct ELISA method.

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