( eled anti-mouse IgG and as ...)A Mix-and-Read Cell-Based Assay for Hybridoma Screening Using the FMAT ,,, 8100 HTS System

eled anti-mouse IgG, and as little as 5 L of hybridoma supernatant. A total of 51 positive wells from 14 plates were easily and unambiguously identified by the FMAT system using a simple mixand- read assay. A cell ELISA screen performed simultaneously with the fluorometric microvolume assay generated inconsistent data that was inconclusive. A select number of hybridoma supernatants that were identified as positive by the FMAT system were confirmed by flow cytometry, establishing that the single- step fluorometric microvolume assay is a simple yet robust method for cell-based hybridoma screening.

Methods

FMAT 8100 HTS System Assay Cells expressing the cell surface antigen were grown in tissue culture flasks, collected, washed with screening buffer (PBS, 2% FCS), and resuspended in screening buffer at a concentration of 1x105 cells/mL. FMAT Blue dye-labeled goat antimouse IgG(Fc) was diluted to 0.4 g/mL in screening buffer. To initiate the hybridoma screen, 100 L of cells, 50 L of the labeled anti-mouse antibody and 5 L of hybridoma supernatant were added to each well of 14 FMAT system 96-well plates. No mixing was required. The final volume of 155 L per well contained 10,000 cells and 0.13 g/mL of detection antibody. The plates were scanned after 2 hours of incubation. A well was considered positive if it had a count of over 50 events. A simultaneous screen using control cells (without antigen expression) was performed as described in Figure 1.

Cell ELISA Cells expressing the cell surface antigen were grown in tissue culture flasks, collected, and plated in 14 96-well plates. After incubating for 48 hours, the tissue culture supernatant was removed and the
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