Renewable, well-characterized antibodies
aSYCP3 – green, aCBX5 – red, DNA - blue
Antibodies are among the most commonly used research tools in biomedicine, and are used to detect proteins; to monitor protein localization in cells and tissues; and to study protein-protein interaction and signaling networks. Unfortunately, high quality antibodies are not available for most human proteins, and this severely limits our ability to explore human biology and understand disease. Partly as a result of this lack of reagents, most biological research remains focused on genes and proteins that were known prior to the sequencing of the human genome ( Edwards et al, Nature). It is generally agreed that exploration of novel proteins depends on the availability of high-quality, renewable antibodies.
Project objectives and scope
The specific goal of this project is to generate and characterize 200 high-quality renewable antibodies to proteins implicated in epigenetic and chromatin biology. The project is being supported by the SGC funders and Life Technologies, which is distributing the antibody reagents with no restriction on use ( epigenetic recombinant antibodies). The renewable antibodies (Fabs or scFvs) are being selected using phage display technologies, using "crystallography-grade" antigens provided by the SGC. The objective is to provide the scientific community the research tools to better understand the roles and functions of the targeted proteins in health and disease.
Antibody generation - background
This large-scale project is based on a pilot project carried out a couple of years ago (Colwill et al, 2011) and is addressing three major challenges associated with creating renewable antibodies for large numbers of human proteins: 1) the availability of high-quality antigens; 2) the need to manage the process effectively: and 3) the lack of generally-accepted quality-control criteria ( Blow Nature 2007; Schofield et al. Genome Biol 2007). The project's "target product profile" is an antibody (either a Fabs or a scFvs) with <20nM affinity for its antigen and that is proven to immunoprecipitate the cognate (endogenous) antigen from human cells. Most antibodies will also be tested for efficacy in chromatin immunoprecipitation and immunofluorescence. Up to two antibodies per protein target are acceptable, provided the antibodies recognize different epitopes on the target protein, and/or are validated for different applications.
The project is being directed by Dr. Susanne Gräslund, Principal Investigator of Biotechnology at the SGC in Toronto, and involves a close collaboration between the SGC, the laboratories of Tony Kossiakoff and Shohei Koide at the University of Chicago, the laboratories of Sachdev Sidhu, Lori Frappier, Jack Greenblatt at the University of Toronto, the laboratory of Stefan Dubel at Technische Universität Braunschweig, and Life Technologies.
Crystallization-grade domains of the target proteins are produced at the SGC, with a protein tag that is biotinylated in vivo. The biotinylated antigens are distributed to the recombinant antibody collaborators, who screen and isolate candidate Fabs or scFvs. Within these laboratories, the candidate Fabs are subjected to primary validation using different types of ELISAs, including counter-selection against another antigen from the same protein family. For the Fab pipeline, the cDNAs encoding the most promising candidates for each targeted domain are sent to the SGC in Toronto where they are subcloned into an expression vector for production in E.coli. The Fabs are then produced in as biotinylated proteins and purified to homogeneity. In some cases, these Fabs are converted to IgGs by fusing the human Fabs with a Mouse IgG1 Fc, and the resulting IgGs are expressed and purified from HEK293F cells. For scFvs the candidate antibodies are converted to IgG-like molecules within the Dubel laboratory and sent to Toronto for validation in cell-based assays.
The purified candidate antibodies (Fabs, IgGs or scFv-Fc fusions) are tested in cell-based assays using the following methods:
- Immunoprecipitation from HEK293 cells over-expressing an FLAG-tagged clone of the full-length target antigen
- The Fab, IgG or scFv-Fc fusion is deemed validated if it immunoprecipitates the FLAG-tagged antigen, as assessed by Western Blot (IP-WB) for the FLAG-tagged protein in the immunoprecipitate (using anti-FLAG antibody)
- Immunoprecipitation of endogenous antigen from HEK293 cells
- The immunoprecipitated proteins are subjected to tryptic digestion and Mass spectrometry (IP-MS) and the mass spectrum assessed for the presence of "signature" peptides from the antigen.
- Activity under Chromatin Immunoprecipitation (ChIP) conditions
- HEK293 expressing the FLAG-tagged antigen are processed as for ChIP and the immunoprecipitaions are resolved by gel electrophoresis and the presence of the antigen detected by Western Blot (using anti-FLAG antibody)
- Immunofluorescence in HEK293 cells over-expressing an FLAG-tagged clone of the full-length target antigen
- The Fab, IgG or scFv-Fc is tested in IF using cell lines over-expressing the target antigens and compared to staining with anti-FLAG antibody as well as RNAi knockdown controls.
- Western blot (WB)
- Using cell lines overexpressing the target antigens.
At minimum our antibodies are validated to work in IP, and we are finding that most also ChIP-compatible and good for IF. Because the antibodies are selected against native (folded) antigens, in most cases they do not recognize denatured protein and thus are not useful for Western Blots.
Different types of renewable antibodies
Hybrid IgG molecule with human Fab part and Fc from mouse IgG1
The project produces three types of antibodies; biotinylated Fabs, single-chain Fv-Fc fusions, and human/mouse IgG1s:
- Fabs are in vivo biotinylated during production by an Avi tag on the heavy chain. These are fully compatible with immunoprecipitation and immunofluorescence (Colwill et al, 2011). Any streptavidin conjugate can be used as secondary reagent for detection.
- scFv-Fc fusions have a scFv part of human origin fused to a human IgG1 Fc part. Any anti-human secondary antibody can be used for detection.
- The recombinant IgGs comprise a Fab part of human origin fused to the Fc part from mouse IgG1. Any anti-mouse secondary antibody can be used for detection.
The renewable antibodies that have been demonstrated to be active for immunoprecipitation can be obtained from Thermo Fisher, along with the validation data.
We encourage all users of the antibodies to visit www.1DegreeBio.com and share your experiences with these reagents with other scientists.