Abstract 5774: Novel cell-based high-throughput hybridoma screening method using the Celigo image cytometer for antibody discovery

Abstract

Abstract Hybridoma screening is a highly important process for antibody discovery, which can identify potential clones from hundreds to thousands of hybridoma cultures against the desired target antigen. Traditional screening methods using ELISA and flow cytometry presented technical issues such as limited accuracy, reduced high-throughput capability, increased time and cost. Conventional ELISA screening with coated antigen can also be impractical for difficult to express hydrophobic membrane antigens or multi-chain protein complexes. Here, we demonstrate novel cell-based high-throughput screening methodology using plate-based Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression. The image cytometry-based high-throughput screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on Chinese hamster ovary (CHO) cells. Both target CHO and CFSE-stained wild type were co-cultured to simultaneously detect target antibodies (Alexa Fluor 594) and nonspecific binding, which can eliminate the need to produce a separate control sample for each hybridoma supernatant. Next, the image cytometer was used to screen 672 unique hybridoma supernatants to develop the high-throughput screening workflow. The Celigo was used to quickly image and analyze antibody binding of 672 samples, using Hoechst, Alexa Fluor 594 and CFSE fluorescence to identify high, medium, and low binding hits. Finally, the sensitivity of the image cytometer was demonstrated by serial dilution of purified CD39 antibody. Celigo was used to measure antibody affinities of commercial and in-house antibodies to membrane-bound CD39. This cell-based screening procedure can be completely accomplished within one day, significantly improving throughput and efficiency of hybridoma screening. Furthermore, measuring direct antibody binding to living cells eliminated both false positive and false negative hits. The image cytometry method was highly sensitive and versatile, and could detect positive antibody in supernatants at concentrations as low as ~5 ng/mL, with concurrent Kd binding affinity coefficient determination. We propose that this screening method will greatly improve screening technologies and facilitate more efficient antibody discovery. Citation Format: Leo L. Chan, Haohai Zhang, William Rice, Nasim Kassam, Maria S. Longhi, Haitao Zhao, Simon C. Robson, Wenda Gao, Yan Wu. Novel cell-based high-throughput hybridoma screening method using the Celigo image cytometer for antibody discovery [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 5774.