Abstract 4035: High throughput validation of antibodies for cancer research
Authors: Shulewitz Mark , Hamilton Tim , Schwartz Kaiti , Thornton Christine , Uy Gerald , Archarya Poulomi , Bogoev Roumen , Wang Yan 
Published: July 2016

Abstract
Antibodies are critical reagents both to study and treat a wide range of cancers. In addition to the relevance of antibodies in immunotherapy, their use in understanding cancer pathophysiology is one of the fastest growing applications. Such applications create a constant demand for high quality antibodies that can recognize a wide range of expression of a specific target that is involved in cancer and metastasis. Even though there are thousands of antibodies available, including those from commercial sources, only a small percentage is well characterized and demonstrated to be of dependable quality.
Establishing the ground rules for rigorous validation of antibodies used for immunodetection of cancer targets is critical for obtaining high quality data. In fact, a number of high profile preclinical study failures have been attributed to the use of inadequately characterized antibodies exhibiting non-specific binding or high background. While researchers typically rely on publications in peer-reviewed journals as absolute indicators of antibody quality, this approach has not proven to be very reliable.
In this poster we describe a robust and high throughput process for validating antibodies for use in western blot applications for cancer research. Some key cancer-related target proteins are detected by antibodies and the validation process is illustrated using these western blot data. In a nutshell, each antibody is screened against an entire panel of lysates from different cells expressing endogenous levels of the target protein. A rapid membrane transfer system coupled with a novel stain-free gel imaging technology is used to ensure a high efficiency of transfer and assess protein quality. Antibody sensitivity and binding specificity is determined based on quantitative data generated using an image analysis software. This software allows us to calculate the ratio of specific to non-specific binding accounting for background within the same lane from the western blot image. We have developed analytical parameters that, when applied to these data, allow us to identify high performing antibodies. The basic principles of this validation method are widely applicable for different types of antibodies and sample types. Overall, this approach ensures the selection of high-performing antibody reagents, thus enabling scientists to get dependable data for academic or preclinical research which, in turn, could significantly advance our understanding of cancer and help develop novel immunotherapies for cancer treatment.
Citation Format: Mark Shulewitz, Tim Hamilton, Kaiti Schwartz, Christine Thornton, Gerald Uy, Poulomi Archarya, Roumen Bogoev, Yan Wang. High throughput validation of antibodies for cancer research. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4035.