Assessing Monoclonality: Statistics
Clonality of hybridomas collected from semisolid medium was previously evaluated and it was concluded that, at appropriate seeding density, the probability of coincidence (nonclonality) is 4%. Genetix has also generated a statistical calculation that shows a correlation between the probability of monoclonality and seeding density/size of colony.
In a typical experiment, CHO cells are plated into semisolid medium to produce 25 colony outgrowths per 35 mm diameter well (i.e., a six-well microplate well). At day 14, the colonies are around 0.75mm in diameter. Using the formula (0.25 x Pi x (0.75+0.75)2 x (n-1)) / (0.25 x Pi x 352), where colony number n=25, the probability of coincidence is 4.4%, thus the probability of monoclonality for one round of cloning should be 95.6%. This is very close to the value determined previously.
Assessing Monoclonality: Experimentation
To test the statistical theory experimentally, we combined two IgG-secreting hybridoma cell lines, one secreting IgG1 and the other secreting IgG2a. Colonies were grown in CloneMatrix-based semi-solid media in the presence of fluorescent-labeled isotype-specific antibodies against the IgG1 and IgG2a. AlexaFluor 488-conjugated anti-IgG1 was visualized in the FITC channel of ClonePix FL, and AlexaFluor546-conjugated anti-IgG2a was visualized in the rhodamine channel. Representative images are shown in Figure 3.
The colonies were analyzed using ClonePix FL software and differentially picked according to whether they were shown to have high exterior mean intensity of FITC with low Rhodamine intensity (for IgG1 secretors), or low FITC with high Rhodamine (for IgG2a secretors).
A total of 312 colonies (156 of each type) were picked and subsequently grown in 96-well plates for four days. The conditioned media were then assessed for IgG1 and IgG2a using isotype-specific ELISA assays (Bethyl Laboratories).
Cells with either poor outgrowth or a negative ELISA for both IgG1 and IgG2a were excluded. The results showed that, of the 143 colonies that were picked based on IgG1-specific fluorescence, only one showed a low level of IgG2a (clonality=99.3%). Of 81 IgG2a colonies picked based on IgG2a-specific fluorescence, three were heterogeneous (clonality=96.4%). Monoclonality for the complete data set was calculated as 98.25%.
This experimental data validates the statistical evaluation that clonality after a single round of picking on the ClonePix FL system is >95.6% when picking larger-sized colonies (0.75mm diameter). Using the same formula, it can be calculated that by picking at an earlier time point (when colonies are only 0.35 mm in diameter), the probability of monoclonality is increased to 99.0%. In addition, performing a second round of cloning (replating the picked clones and repicking) increases the probability of monoclonality to 99.9%.
The ClonePix FL system has been demonstrated as a powerful tool for rapid screening and isolation of secretory mammalian cell lines typically used in the production of therapeutic proteins and research monoclonal antibodies. The system increases overall productivity, leaving more time to better characterize antibodies and begin new projects. The data presented in this tutorial supports the idea that picking colonies of mammalian cells from semisolid medium is an effective means to isolate clonal cell lines.