Response letter

In the letter “A cardinal sin when researching neuropsin/ KLK8: Thou shalt validate antibodies,” Lahiri et al. [1] respond to a study published last December by Herring et al. in Alzheimer’s & Dementia [2]. More specifically, they criticize Herring et al. for an apparent “lack of information on antibody validation”; in particular, they question the antibody against neuropsin (syn. Kallikrein 8, KLK8)—a commercially obtained antibody used also in many other studies. By the term “validation” they do not mean the standard steps, such as, showing a distinct band with a specific molecular weight or no bands when omitting the secondary antibody, but they mean five approaches for antibody validation recommended in an article published recently in Nature Methods 2016;13:823–7. These approaches are as follows: First, to eliminate the expression of a target protein by genome editing or RNA interference and subsequently prove an elimination of antibody labeling. Second, to perform a parallel antibody-independent analysis, such as mass spectrometry. Third, to use an independent antibody with a nonoverlapping epitope. Fourth, to tag the targeted protein, for example, by green fluorescent protein and show a significant correlation between the amount of antibody labeling and the detection of the epitope tag. And fifth, to capture theWestern blot bands and perform mass spectrometry. These are indeed commendable recommendations, but I am not aware of a single study in which a commercially obtained antibodywas validated according to them before its application for further experiments. In fact, when using 20 or more antibodies in a single study it would be economically unviable and extremely tedious, if each working group were to validate a purchased antibody in this manner. These recommendations can only be seen as a proposition or appeal to the manufacturers of these antibodies. Of course, Lahiri et al. have every right to voice a different opinion and demand a validation from the final user each time. However, it is somewhat arbitrary that this specific study by Herring et al. is the focus of their scrutiny. As they themselves state in their letter: “This is related to a problem that is pandemic in molecular biology (including proteins) research..” If this is the case, would it not be more judicious to formulate a general letter of appeal and direct it to everyone who performs such protein quantifications?