The enzyme-linked immunosorbent assay (ELISA) uses antibodies and a solid-phase enzyme immunoassay to detect the presence of a specific antigen in a liquid or wet sample. Antigens from the sample are immobilised on a solid support either non-specifically or specifically. A specific antibody is then applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme or can itself be detected by an enzyme-linked secondary antibody. In the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a visible signal, most commonly a colour change in the substrate, which indicates the quantity of antigen in the sample. When the presence of an antigen is analysed, the name "direct ELISA" refers to an ELISA in which only a labelled primary antibody is used, whereas the term "indirect ELISA" refers to an ELISA in which the antigen is bound by the primary antibody which then is detected by a labelled secondary antibody.

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Protocol Series:
Indirect ELISA
Information on the general steps required for carrying out Indirect ELISA
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2. The enzyme-linked immunosorbent assay (ELISA) uses antibodies and a solid-phase enzyme immunoassay to detect the
presence of a specific antigen in a liquid or wet sample. Antigens from the sample are immobilised on a solid support
either non-specifically or specifically. A specific antibody is then applied over the surface so it can bind to the antigen.
This antibody is linked to an enzyme or can itself be detected by an enzyme-linked secondary antibody. In the final step,
a substance containing the enzyme's substrate is added. The subsequent reaction produces a visible signal, most
commonly a colour change in the substrate, which indicates the quantity of antigen in the sample.
When the presence of an antigen is analysed, the name "direct ELISA" refers to an ELISA in which only a labelled
primary antibody is used, whereas the term "indirect ELISA" refers to an ELISA in which the antigen is bound by the
primary antibody which then is detected by a labelled secondary antibody
The following slides provide information on the general steps involved. Optimisation may be required.
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3. Indirect ELISA
1. A buffered solution of the antigen to be tested for is added to each well of a microtiter plate, where it is
given time to adhere to the plastic through charge interactions.
2. A solution of non-reacting protein, such as bovine serum albumin (BSA) or casein, is added to the wells
(usually 96-well plates) in order to cover any plastic surface in the well which remains uncoated by the
antigen.
3. The enzyme-conjugated primary antibody is added, which binds specifically to the test antigen coating the
well.
4. The enzyme-conjugated secondary antibody, specific to the primary antibody, is added.
5. A substrate for this enzyme is then added. Often, this substrate changes colour upon reaction with the
enzyme.
6. The higher the concentration of the primary antibody present in the serum, the stronger the colour
change.
7. A spectrometer is used to give quantitative values for colour strength.
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