Optimization for Testing the Sensitivity and Specificity of Antibodies to Brg1 - Abstract
Antibodies are vital experimental tools that are used extensively in basic science and in clinical settings. With the evolution of personalized medicine and targeted therapies, the development of reliable diagnostic antibodies will be in greater demand, as they are used for a variety of assays such as immunocytochemistry (ICC), immunohistochemistry (IHC), immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), western blot (WB) and immunoprecipitation (IP), among others. While antibodies are largely produced commercially, the performance standards are most often defined by individual investigators who use an antibody for specific assays. Central to the reliability and validity is to what degree the antibody has been produced and tested to assure its specificity and selectivity. This, in turn, often requires specific scientific expertise and knowledge which often lie with individual investigators and not necessarily with commercial companies. Specifically, detailed knowledge is required to remove possible cross-reactive proteins which results from multiple
epitopes with a similar sequence or with an overlapping sequence(s); these antigens can be easily eliminated with a well-designed double immunopurification step. Often, multiple antibodies are commercially available such that the decision becomes more complicated as one has to determine which of the antibodies are sensitive and not cross-reactive. Hence, it is sometimes difficult for investigators to find the “right” antibodies. The generation of antibodies against the SWI/SNF subunit BRG1 (Brahmarelated gene 1, SMARCA4), which has a high sequence homology to its counterpart BRM (Brahma, SMARCA2) represents such a paradigm. Within this article, we discuss a series of steps that scientists, pathologists and technicians can use to identify the “right” antibody for their purposes. Using a set of commercially-available antibodies to BRG, we detail a cadre of steps to assure that the chosen antibody possesses the prerequisite sensitivity and specificity. This is important as many companies do not rigorously test and perform thorough work-ups of their antibodies.