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

wells were blocked with 200 L of screening buffer for 1 hour. The buffer was removed and replaced with 5 L of hybridoma supernatant and 95 L of fresh buffer. After 1 hour of incubation, the supernatant was removed and the wells were gently washed 6 times with buffer. Detection antibody (goat anti-mouse IgG (Fc)-HRP) was added, followed by an additional hour of incubation. The supernatants were removed, the wells were gently washed 6 times with buffer and substrate was added. The color was allowed to develop for 5 to 10 minutes before stopping reagent was added. Plates were read using a standard plate reader.

Results

Out of a total of 1,344 wells, 51 were considered positive by the FMAT 8100 HTS System. The positive wells were further subdivided into those with low (< 500: 32 wells), medium (500-1,000: 14 wells), or high (>1,000: 5 wells) average fluorescence intensity (FL1) values.

A simultaneous screen using control cells that do not express cell surface antigen did not yield any hits. The FMAT software allows the results to be viewed in several different formats (Figure 1, panels A-D) for simple and unambiguous selection of positive wells. In addition, a tab-delineated file containing a summary of the data from all fourteen plates and in 96-well grid format can be accessed. A detailed well view allows for real-time or postscan evaluation of the positive wells (Figure 2). A cell ELISA screen was performed in parallel in an attempt to confirm the data obtained with the FMAT system. However, the data from the cell ELISA was inconclusive, prohibiting any meaningful comparisons. For example, the FMAT system easily identified 6 positive wells from one plate of the screen (Figure 3, panel A). However, only one positive well i
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