I am a manager of a monoclonal Mab core lab for the university of Colorado, we have a history of successful hybridoma production but recently we have had problems with getting hybridomas from a mouse that shows a strong signal. On the first screening we will get strong positives that would traditional result in good hybridomas, but recently I have seen the positives stop producing the Mab after the initial screen. Few make it to cloning but don’t produce Mab after that. Do you have any idea why this is happening? We’re using mice that have had at least 3-4 injections with the protein of interest and are screened by Elisa titration assay and by a western. A traditional fusion is done with PEG to SP2/0 cells and plated into 96well plates. They are normally screened in 7-10 days. Media is IMDM+15% FBS +HAT


Thanks for your question and providing all the details. I think you are writing that this is happening with one animal and not all the animals you are recently harvesting cells from. Assuming this is the case, I think you got a bad mouse . You also mentioned that spleen cells from the animal initially produce antibody but then production stops even before fusion occurs. It just might be that the cells you harvested from this one animal are more unstable than normal. Was this an older mouse? Making spleen cells, and fusing them do create hybridoma’s creates non-normal cells and many cells are lost or never grow because fusion is lethal. Hybridoma cells that survive run a range of genetics and have abnormal chromosomes anyway. Maybe your cells rearrange more than normal. It is always suggested that you do a clonal isolation of cells as soon as possible after selection This will minimize the variation in your culture of cells. If you took at some of those hybridoma cells in the 96 well dish that were producing antibody, but now are not,and serial cloned them, I bet some of them are doing what you want. You don’t see the productivity since you are sort of diluting out the signal. Try cloning some cells since it is better than starting over with a new mouse. Also if you do single cell cloning make sure to use conditioned medium initially when you first get the one cell in a well.

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