Category: Assay Development and Screening
Monoclonal antibodies are the fastest-growing class of therapeutic agents with success in treating a wide range of diseases. Most therapeutic antibody candidates are initially generated from mouse hybridomas or primary B cell clones after antigen immunization. Identifying clones that secrete sufficient levels of mouse IgG is critical to qualify the stable clones for downstream antibody gene cloning. Traditionally, screening culture supernatants for mouse IgG levels is done by ELISA, followed by separate IgG isotyping, cell counting and viability assays. We developed a no-wash, multiplexed high throughput flow cytometry screening assay that simultaneously provides IgG concentration, IgG isotype, cell count and cell viability information in each well of multiple microtiter screening plates.
We validated this assay by screening mouse hybridoma samples and primary B cell samples in a 96-well plate format. The Panorama feature of the ForeCyt 6.1 software allows rapid multi-plate comparison and combined with the profile map algorithm, generated multiplexed readouts for each well with these data: IgG concentration, IgG isotype, cell density and cell viability. Boolean logic-driven profile maps identified wells meeting multiple IgG isotype concentration criteria. These user defined thresholds identified pure monoclonal hits, while excluding samples containing multiple IgG isotypes within the same well.
ForeCyt Panorama algorithm was applied to our data set to alleviate a common problem in multi-plate screening campaigns in which hit criteria used for one plate may not be applicable to other plates due to plate-to-plate variations. The Panorama feature creates a comprehensive data visualization in a single working space that displayed plate-based data metrics including: plate design, heat maps, profile maps, series and line graphs and data grids for the entire multi plate screening study. Heat maps display color spectrum scaling between the min/max range of all plates, individual plate range or user a defined range. This capability enables global or local visualization of each data metric for all plates. Profile maps in the Panorama view have criteria slider bars in which user defined criteria can be changed to identify mouse monoclonal hits globally across all plates in real-time. Monoclonal hits identified across the plates using profile maps allowed us to then generate a line graph to compare multiple important hybridoma metrics and to display the ranking of mouse hybridoma or B cell hits based on user-defined specifications.
The Panorama function with profile map algorithm in ForeCyt6.1 software is an intuitive tool that eliminates multiple steps and provides the “big picture” data visualization that guides the accurate decision-making process essential for selecting stable mouse antibody clones with high IgG secretion, single IgG isotype and optimal cell health.
Keywords: monoclonal antibody discovery, mouse IgG isotype, high throughput screening, multi-plate data analysis, multiplex assay
Presentation Format: Poster; Scientific Track: Assay Development and Screening Track