how to improve hybridoma production? - (Jun/03/2011 )

Dear colleagues,

I wonder if any of you have succeeded in improving the fusion protocol to get a significantly higher number of antibody-producing hybridomas than following standard procedures.
For instance, according to the literature you can improve hybridoma yield by first freezing the lymphocytes. In another paper, it is claimed that preincubation of cells in 0.25% PEG prior to fusion improves hybridoma number too. Still according to a third paper, adding pre-chilled PEG to the cells on ice also improves results.
The point is that I have tried all these variants without getting impresive results, not even significant changes.
Can you tell me about your particular experience?
Thanks a lot!

Chelo on Fri Jun 3 10:26:02 2011 said:

Have you tested cloning efficiency with your particular batch of FBS? Serum lots vary in their ability to support hybridomas at low density (often by >10 fold). We always test a sample of a new serum batch before we buy it. Simply plate out a hybridoma (any will do) at ca. 1 cell/well in a 96 well microplate. After 8-9 days of incubation AT LEAST 40 wells should show signs of growth. Poor batches, from our supplier about 75 % of those tested, show growth growth in < 10 wells.
Thus, by selecting for good batches of serum you can dramatically increase the number of primary hybrids.

Hope this helps.

klinmed on Mon Jun 6 11:28:39 2011 said:

Dear Klinmed,

thank you very much for your idea. Actually, I didn´t know FBS batches of a given brand could be so variable. However, my problem is not that I have variable results from fusion to fusion, it is only that the number of hybridomas is very low compared to the number of cells used for fusion. I am looking for protocol "tricks" that could increase >10 fold the hybridoma yield.

Chelo on Mon Jun 6 11:58:35 2011 said:

Hi again,
I think that you may have missunderstood my reply to your post.
If you divide cells from the SAME fusion step into two sets of microplates, one containing medium made with "good" serum, and another set with "bad" serum, you will notice dramatic differences in the yield of primary hybrids obtained with each of the serum batches. Thus, merely by pre-selecting FBS you can dramatically increase your hybrid yield.
Another possibility for your low yields is that the mice are not producing a strong enough immune response. Do you test-bleed the animal before fusion (antibody titer should be at the very least >1:500)?
Using 5E7 splenocytes and 2.5E7 NS0 myelomas we almost invariably get growth in every well of the the 20 96-well microplates (yield >2000 hybridomas) we usually seed with the fused cells.