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 that can identify potential clones from 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. Here, we demonstrate a novel cell-based high-throughput screening method using the 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 screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on CHO cells. Both target CHO and CFSE-stained wild type were co-cultured to simultaneously detect target antibodies 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 hybridoma supernatants to develop the screening workflow. The Celigo was used to quickly image and analyze antibody binding using Hoechst, Alexa Fluor 594 and CFSE fluorescence to identify high, medium, and low binding hits. In addition, the Celigo was used to measure the binding affinities of the target antibodies to membrane-bound CD39. 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. We propose that this method will greatly improve screening technologies and facilitate more efficient antibody discovery.