Isotype Control Function & Applications
Immunoassay techniques such as Flow Cytometry, ELISA or Western Blot provide scientists with an excellent way to detect whether a sample contains a target particle, like protein, peptide or hormone. This is achieved thanks to antibodies ability to bind to antigens. This hook-like mechanism allows for scientists to 'fish out' whatever particle they desire. However, it is important to set up controls in such experiments, as antibodies can bind to other undesired molecules. Thus it is important to control such nonspecific binding, to ensure that your detection experiments run smoothly. This is achieved through isotype controls.
Do note that isotype controls are primarily used in flow cytometry, but can also be utilized in other applications like western blotting and ELISA.
Negative controls work by not giving a response, which is to be expected. In not having a result, scientists can have greater confidence that their experiment is working as it should. The isotype control is one such tool, and work by being a copy of the primary antibody used in the test, but lack specificity. That is, an isotype control is a primary antibody with the same Fc-region as the actual experiment but a different variable region, thus it has a non-specific binding region. More specifically, the isotype control antibody should be the same isotype of heavy chain and light chain, the same fluorchrome and F:P ratio. This enables scientists to confirm the specificity of binding in the experiment. Simply put, to show that the used antibody is specific, these isotype controls should not result in similar staining or band patterns.