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

the many wash steps of the procedure is a significant source of variability in a cell ELISA. An alternative to a cell ELISA is flow cytometry, which detects cell-surface staining by specific antibodies. However, flow cytometric analysis routinely involves handling and/or analyzing each sample individually in addition to numerous wash and incubation steps. Since thousands of clones are routinely screened, flow cytometry is not a practical option for high-throughput hybridoma screening. An ideal assay for hybridoma screening would be one that combines the reliability of flow cytometry with the plate format of a cell ELISA and can be performed without wash steps. The FMAT 8100 HTS System provides a unique platform that is perfectly suited for hybridoma screening. The FMAT system is a macroconfocal scanner that visualizes and quantitates both cell- and bead-bound fluorescence. Light generated from a 633 nm helium/neon laser scans an area of 1 mm2 with a depth of focus of ~100 m from the bottom of each well. Because the depth of focus is relatively small with respect to the remaining volume of the well, the majority of the unbound fluorophores remains undetected and need not be washed away. The FMAT software is then able to subtract the remaining background fluroscence from the fluorescence immobilized on the cells or beads. As a result, all assays performed with the FMAT system are mix-and-read and do not require wash steps. The integrated plate handler allows for walk-away screening for up to sixty 96- or 384- well plates. A screen for a monoclonal antibody directed against a cell-surface antigen was developed using the FMAT system. Each well contained cells expressing the cell-surface antigen of interest, FMAT Blue dye-lab
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