Implementation of an antibody validation procedure: Application to the major ALS/FTD disease gene C9ORF72

Abstract

Rigorously characterized antibodies are a key resource in biomedical research, yet there are no community-accepted processes to characterize the quality of research grade antibodies. This has led to a proliferation of poorly characterized antibodies of suspect quality, which in turn has led to flaws in the literature that hamper research progress, including the study of human disease. We selected the human protein C9ORF72 as a test case to implement a more standardized antibody characterization process. Mutations in the gene C9ORF72 are the major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia but much remains unknown regarding the function of the encoded protein. We used CRISPR/Cas9-based knockout cells and knockout mice as controls to characterize 14 C9ORF72 antibodies including 12 commercially available antibodies advertised as selective for C9ORF72. We found one monoclonal antibody that is specific for the protein in immunoblot and that also recognizes C9ORF72 specifically in immunohistochemical applications on brain sections. A second C9ORF72 monoclonal antibody is effective for immunoprecipitation and immunofluorescence. Some of the antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over conclusions regarding previously reported C9ORF72 properties.