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Indirect and Sandwich ELISA
Monoclonal and polyclonal antibodies can be used to detect and quantify the presence of proteins in a method called enzyme-linked immunosorbent assay (ELISA). Although there are many variations of this method, two important ones are the indirect ELISA and sandwich ELISA. Indirect ELISA is used to test for the presence of some specific antibody. In this method, a well is first coated with the antigen that the antibody will bind to. A sample of proteins is then added into the well. If the antibody of interest is present, it will bind onto the antigen on the surface of the well. A second enzyme-linked antibody is added. This enzyme-linked antibody can bind onto the antibody-antigen complex. Once bound, any other impurities remaining in the solution are washed away. In the final step, the substrate for the enzyme attached onto the antibody is then added and this causes a color change in the solution. This color change signifies the presence of the antibody of interest. The darker this color change is, the higher the concentration of antibodies in our solution is. In sandwich ELISA, the entire point is to test for an antigen rather than an antibody. In this method, we coat the bottom of the well with an antibody that can bind the antigen of interest. We then add a sample that might contain the antigen. If the antigen is present, the antibody will bind the antigen. In the next step, the enzyme linked antibody is added, which forms a "sandwich" antibody-antigen-antibody complex. Once the solution is washed and the substrate is added, a color change takes place as long as the antigen is present.
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