Direct ELISA Protocol

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.

1. www.stjohnslabs.com Protocol Series: Direct ELISA Information on the general steps required for carrying out Direct ELISA St John's Laboratory Ltd St John's Laboratory Ltd @ StJohnsLabs St John's Laboratory Ltd For more in our protocol series, visit Slideshare.net/ StJohnsLabs

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 . www.stjohnslabs.com

3. Direct ELISA 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. 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. The enzyme-conjugated primary antibody is added, which binds specifically to the test antigen coating the well. A substrate for this enzyme is then added. Often, this substrate changes colour upon reaction with the enzyme. The higher the concentration of the primary antibody present in the serum, the stronger the colour change. A spectrometer is used to give quantitative values for colour strength. www.stjohnslabs.com

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