Hybridoma screening is a critical step for antibody discovery, which necessitates prompt identiﬁcation of potential clones from hundreds to thousands of hybridoma cultures against the desired immunogen. Technical issues associated with ELISA- and ﬂow cytometry-based screening limit accuracy and diminish high-throughput capability, increasing time and cost. Conventional ELISA screening with coated antigen is also impractical for difﬁcult-to-express hydrophobic membrane antigens or multi-chain protein complexes. Here, we demonstrate novel high-throughput screening methodology employing 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-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. Next, 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 improv-ing 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 facilitate antibody discovery and screening technologies.
Example ﬂuorescence images and intensity scatter plots for AF594 and CFSE at different dilutions of the primary antibodies. CD39-expressing CHO cell population showed decrease in ﬂuorescence signals as the dilution factor increased.
The above work has been published in the Journal of Immunological Methods. Zhang et al. Novel high-throughput cell-based hybridoma screening methodology using the Celigo Image Cytometer, (2017) J Immunol Methods. 447: 23–30. https://www.ncbi.nlm.nih.gov/pubmed/28414024