Abstract 5763: Large-scale use of knockout validation to confirm antibody specificity to key oncology targets

Abstract

Abstract Antibodies are the most common tools in basic science and clinical research. Over the past several years there has been an ongoing discussion around antibody validation in light of poor experimental reproducibility between laboratories. This is in part due to the cross-reactivity between antibodies and off-target proteins and the variability between different antibody batches. As a result, experimental irreproducibility leads to wasted resources and compromises the advancement of science. In response to this, Abcam has introduced knockout (KO) validation to help produce target-specific antibodies. This initiative uses KO cells lines made possible through a partnership with Horizon Discovery. Target genes are mutated via CRISPR-Cas9 within a haploid cell line. This results in a frameshift and a complete loss of gene expression. These KO cell lines provide a true negative control for antibody validation as a lack of signal confirms that the antibody binds to its intended target. As a result, both antibody specificity and reproducibility on a large scale can be addressed. We have KO-validated over a thousand antibodies, including several that are relevant to immuno-oncology research. Examples of such targets are the ERK1/2 pathway, which regulates cell survival, proliferation and cell death. Inhibition of this pathway is an attractive target for the treatment of malignant tumors with increased ERK1/2 activity. Similarly, Ki67 expression is strongly associated with tumor cell proliferation and can be used as a marker for tumor aggressiveness. Finally, overexpression of vimentin in tumors correlates with increased growth and invasion, leading to poor prognosis. It is imperative that reproducible data can be produced on these targets to further our understanding of tumor biology and patient prognosis. Here we present the data for antibodies to these key targets that have been validated in both flow cytometry and immunocytochemistry. The antibodies have been tested in KO and wild-type cells to confirm their specificity. The antibodies shown gave signal in wild-type cells but showed complete loss of signal when tested in knockout cell lines. By providing researchers with reliable and specific antibodies that work first time we hope to minimize wasted resources and improve reproducibility. Citation Format: Sam Heaton, Monica Sassi, Alejandra Solache, Bruce Hamilton, Sambhav Dave. Large-scale use of knockout validation to confirm antibody specificity to key oncology targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5763.