Antibodies are key to an ELISA and provide the basis for the specificity and sensitivity of the assay. In a previous blog post, we discussed factors such as antibody affinity, antibody specificity, and antibody titer when choosing a pair of antibodies for ELISA development.
To continue with the antibody selection process, we are going to discuss the various types of ELISAs (antibody capture, antigen capture, and sandwich ELISAs) and the appropriate antibodies for those formats. You can also browse https://www.bosterbio.com/featured-products to know more about antibodies.
Often, the type of antibodies that are available are the limiting factor for which assay can be developed as indicated in the table. Once you've decided on antibodies and the form of assay you will develop, you will need to ensure that the antibodies will work.
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If you've chosen two monoclonal antibodies, you will need to verify that the capture antibody does not change the antigen's immunogenic properties. This could affect the binding of antibodies at other epitopes.
Epitopes of the two monoclonals cannot overlap and must be spatially separated so the binding of one antibody does not compete with the binding of another. To prevent this, known what epitopes each monoclonal recognizes or use a polyclonal.
In a polyclonal-monoclonal sandwich ELISA, often the polyclonal will be used as a capture antibody to pull antigen down and a monoclonal is used as the detection for specificity.
A "self-sandwich" ELISA can sometimes be produced using the same antibody for capture and detection. This will only work for large molecules with many epitopes spaced far apart. Use the antibody with higher affinity as the coating antibody.