Optimize, Adapt and Overcome - 25

In this process, parental hybridoma clones were expanded, followed
by analysis and picking of the desired clones on ClonePix Systems.
Using the FITC-labeled CloneDetect Reagent, clones were imaged and
analyzed for fluorescent intensity using the ClonePix Software. FITC
positive clones were ranked and picked based on the high exterior
mean intensity [FITC] values. Overall, 480 FITC-IgG positive clones
were picked which represent only 5-6% of the total population of
parental clones screened. The majority of high secreting clones were
small (40-60 cells) versus the larger, faster growing clones (70-150
cells) that were determined to be negative IgG secretors (Figure 3).

Figure 3. Detection and selection of viable, high secreting
colonies using ClonePix Software analysis. A: Scatter plot shows
a liner correlation between Exterior and Interior Mean Intensity
suggesting that the IgG is being secreted properly (otherwise
immobilized on the cell surface with high Interior and low Exterior),
and the low IgG yielding clones are due to a heterogenous population
of variable secretors (i.e., only a few hybridomas, 5-6% of the total
population, are producing IgGs, while the majority of the clones
are growing without IgG expression). B: Ranking plot shows clone
selections in purple: the cutoff was FITC signals to pick 480 clones
(Exterior above 700 and Interior above 1,000).

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Optimize, Adapt and Overcome

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