A consensus platform for antibody characterization

Abstract Antibody-based research applications are critical for biological discovery. Yet, there are no industry standards to compare the performance of antibodies in various applications. We describe a knockout cell line-based antibody characterization platform, developed, and approved jointly by industry and academic researchers that enables the systematic comparison of antibody performance in western blot, immunoprecipitation, and immunofluorescence procedures. The scalable protocols consist of (i) the identification of appropriate cell lines for antibody characterization studies, (ii) development/contribution of isogenic knockout controls, validated at the protein level, and (iii) a series of antibody characterization procedures focused on the most common uses of antibodies in research. Guidelines for the assessment of antibody performance are presented along with the value of making the resulting data publicly available. This antibody characterization platform can be implemented with minimal technological limitations. The proposed antibody characterization platform may be performed readily on antibodies targeting a protein in roughly one month, with one person working less than full-time. Antibody characterization is needed to meet standards for resource validation and data reproducibility, increasingly required by journals and funding agencies.