Validation of antibodies: Lessons learned from the Common Fund Protein Capture Reagents Program

Abstract

Large-scale generation of protein capture reagents remains a technical challenge, but their generation is just the beginning. Validation is a critical, iterative process that yields different results for different uses. Independent, community-based validation offers the possibility of transparent data sharing, with use case–specific results made broadly available. This type of resource, which can grow as new validation data are obtained for an expanding group of reagents, provides a community resource that should accompany future reagent-generating efforts. To address a pressing need for antibodies or other reagents that recognize human proteins, the National Institutes of Health Common Fund launched the Protein Capture Reagents Program in 2010 as a pilot to target human transcription factors. Here, we describe lessons learned from this program concerning generation and validation of research reagents, which we believe are generally applicable for future research endeavors working in a similar space.

Fig. 1.. Schematics of the PCRP program.

Generation of human transcription factor (TFs) antigens (as recombinant GST-tagged protein fragments) in bacteria was a high-throughput endeavor. Generation of capture reagents was divided into two groups: as either recombinant antibody, rAb (low throughput) or monoclonal antibody, mAb (high throughput). rAb-ND—not determined as no antibody expression was detected. So, no assay could be done. The reasons for this remain unknown. The mAbs were validated in a medium-throughput fashion by both Western blot and IP with a moderate pass rate. The recent effort by Lai et al. describes a medium-/high-throughput approach to validate around 900 mAbs by ChIP-Exo with a moderate pass rate, and a subset of these were used in ChIP, STORM, CUT&RUN, and PBM assays with a low pass rate. With so many use cases, each community will have to validate for that particular technology and even specific platforms for their own specific purpose.