Successful antibody discovery programs need to rapidly identify and characterize target-reactive candidates. This case study demonstrates how the iQue Advanced Flow Cytometry Platform can be used for target cell line generation, mouse sera evaluation and hybridoma library screening.
Applications for Antibody Screening
Rapid evaluation of clones transfected with a cell surface target antigen. Selection of healthy clones that express high levels of the target antigen ensure optimal performance in downstream hybridoma screens.
Visualization of Screen Results for Generation of Target Cell Line
Results from the screen visualized using ForeCyt. Cells were transduced with lentivirus encoding target antigen, cultured under selection pressure, and sorted into 96 well plates. After cell growth, aliquots of cells were transferred to assay plates, and tested for surface antigen expression using a fluorescently tagged antibody specific to that antigen using the iQue Advanced Flow Cytometry Platform. Cell number was simultaneously measured as an indication of cell growth.
Profile maps show wells that contain high numbers of cells that also express high levels of the target antigen on their surface.
The plate view shows details of the expression levels of the target antigen on the surface of cells. Expression data from untransfected control cells is overlaid to easily visualize positive clones.
Large scale cell based experiment to identify immunized mice producing high levels of antibody against target antigens. Simultaneously testing sera from dozens of immunized mice improves chance of successfully generating hybridomas with optimal antibody reactivity.
Visualization of Screen Results for Mouse Sera Evaluation
Results from the testing visualized using the HeatMap feature in ForeCyt. 3 cell lines were color coded with the fluorescent Cell Encoder dye, mixed together and dispensed into wells of 384 well plates. Serum samples were added to each well, followed by a fluorescent detection antibody and plates were analyzed using the iQue Advanced Flow Cytometry Platform. Sera from 36 mice pre-immunization and post-immunization with the target antigen were tested in duplicate in 8 fold dilutions.
Heatmaps show results for antibody binding to control cells, cells expressing target antigen and cells expressing a related but irrelevant antigen.
A high throughput, multiplexed screen that simultaneously tests for binding to target antigens but not control antigens. The entire screen and data analysis were completed in one day.
Visualization of Screen Results for Hybridoma Library Screening
Results from the entire screen visualized using the Panorama feature in ForeCyt. 3 cell lines were color coded with the fluorescent Cell Encoder dye mixed together and dispensed into wells of 384 well plates. Hybridoma supernatants were added to each well, followed by a fluorescent detection antibody. 9,600 hybridomas were tested from 2 different mice immunized with cells expressing target antigen. The entire screen was completed in 1 day.
Heat maps (red) showing results for antibody binding to control cells, cells expressing target antigen and cells expressing a related but irrelevant antigen.
Profile maps (blue) combine the binding results to the three cell types, showing antibodies that bind to cells expressing the target antigen, but not to cells expressing an irrelevant antigen or to negative control cells.
Resources for Antibody Screening
iQue Advanced Flow Cytometry Platform Advantages
Select antibody clones with desired characteristics using the Profile Maps feature on ForeCyt Software.