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A Mix-and-Read Cell-Based Assay for Hybridoma Screening Using the FMAT ,,, 8100 HTS System

s evident from the same plate assayed with a cell ELISA (Figure 3, panel B). The remaining wells identified as positives by the FMAT system are obscured by the high fluorescent background in the cell ELISA plate. Another factor influencing the outcome of the cell ELISA was the high degree of variability in the number of cells per well, resulting in unreliable comparative data. Although great care was taken, cells were nonetheless lost during the many wash steps because of the cells poor adhesion properties. Because no wash steps are required for the fluorometric microvolume assay, variability in the data due to cell loss is not a concern. Since a direct comparison between the FMAT system and the cell ELISA was not feasible, several of the positive wells identified by the FMAT system were analyzed by flow cytometry (data not shown). In all cases, the FMAT system and the flow cytometry data were in agreement with respect to identification and quality of the hits.

An additional advantage of the FMAT system over the cell ELISA is the ability to visually inspect the wells. Images of the positive wells from the same plate represented in Figure 2 are depicted (Figure 3). The fluorescence intensity observed on the cells matched the average FL1 values obtained for those wells. On rare occasions, a false positive may occur if a well contains an abundance of fluorescent debris. By inspecting the images an observer can easily see the debris and remove that well from consideration. Levels of IgG were determined for several of the positive wells and one negative well.

Although some of the wells with high average fluorescence intensity values contain high levels of IgG (e.g. wells 2E11, 6A10), this correlation does not extend to all of the wells (e.g. wells 2A7, 6B5). The quality of the antibody-antigen binding reaction can therefore be eval
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