Successful antibody discovery programs need to rapidly identify and characterize target-reactive candidates. This case study demonstrates how the iQue® High-Throughput Screening (HTS) by Cytometry Platform can be used for target cell line generation, mouse sera evaluation and hybridoma library 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.
400 engineered cell clones screened for high surface expression of target antigen.
5 x 96 well plates run and analyzed in 1 hour.
Multiplex cell based assay enables single-step screening for cell lines that have healthy growth and express high levels of surface antigen.
Use profile maps to visualize clones that express high levels of target antigen and have grown to high cell numbers.
Results from the screen visualized using iQue Forecyt® Software. 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® High-Throughput Screening 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.
Sera from 36 immunized mice tested for binding to cell surface target antigens.
Sera from mice pre- and post-immunization were tested in 8 fold dilutions in duplicate.
Multiplex cell based assay enables single-step screening for sera that bind to target antigens but not other antigens.
Use heat maps to visualize mouse sera that have high titers of antibodies that bind to target antigens but not other antigens.
Results from the testing visualized using the HeatMap feature in iQue Forecyt® Software. 3 cell lines were color coded with the fluorescent Cell Encoding 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® High-Throughput Screening (HTS) by 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.
9600 hybridoma clones screened from 2 different immunized mice.
25 x 384 well plates run and analyzed in 1 day.
Multiplex cell based assay enables single-step screening for antibodies that bind to target antigens but not other antigens.
28,800 data points instantly combined and processed into binding metrics.
53 hits visualized that bind to target but not control antigens.
Results from the entire screen visualized using the Panorama feature in iQue Forecyt® Software. 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.
Select antibody clones with desired characteristics using the Profile Maps feature on iQue Forecyt® Software.
Learn how scientists and ModiQuest Research incorporated the iQue® High-Throughput Screening (HTS) by Cytometry Platform across different stages of th...
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Enable real-time, label-free analysis for the determination of kinetics, affinity and antibody/protein quantitation.
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