We are having problems with the generations of mouse monoclonal antibodies (MAbs). We have routinely produced MAbs before, but lately started having problems. Basically, we have hybridomas in more than 90% of the wells 10-15 days after the fusion, while the unfused SP2/0 negative control cells are dead in HAT media by day 5. The hibridomas look normal and grow fast, but they not only do not secrete antigen specific antibodies, they do not secrete antibodies at all. None of the hybridoma sups is positive in an anti-mouse gamma Fc/anti-H+L sandwich ELISA to detect mouse IgG (positive controls, old hybridoma sups and purified mouse MAbs (IgG1) are clearly positives). The test for mycoplasma (Uphoff, C. C.; Drexler, H. G. Detection of mycoplasma contamination in cell cultures by PCR analysis, Human Cell 12:229–236; 1999) was negative for the SP2/0 and hybridomas. I will truly appreciate any advice. Thanks.
Before fusion, how good is your anti-sera? Did you test the Ab titer in the serum?
There are several reasons to meet with this unusual enough phenomenon.
First, check the size of the lymphoid organ used (very often spleen): is it very swollen, synonymous with good lymphoblastic transformation.
Did you have a large number of lymphocytes, synonymous with a significant number of B cells for fusion, and not T cells
Next, verify that the same adjuvant was not injected several times; in the case of the Freund's complete adjuvant that has a dramatic effect with many macrophages.
It is possible that you have only IgMs, but this seems unlikely because the ELISA test used can also assay the IgMs.
A last question concerns the mice used: is it a transgenic mouse or a mouse who is pre tolerization protocol: this can significantly affect the lymphoblastic transformation necessary to achieve secreting clones.
Thanks Jean-Jacques. We work with normal Balb/c mice. Priming with FCA and a couple of boosters with FIA. Serum titles (IgG) are higher than 20K, fusion is performed 4 days after the final booster in PBS. Spleen is typically enlarged (about 40 millon lymphocytes). This problem have been recurrent in the past months, it is not just one experiment. We have changed the source of SP2/0 cells to see if this was the problem, but the result was the same. We are puzzled because we are using the same protocol that we have successfully used for years.
Dear Gualberto,
I think the problem may be the spleen size and timing of the final boost.
4 days after the final boost, blast cells are removed from the spleen, and it remains in majority of T cells, macrophages and granulocytes. The number of total cells confirms this elimination of B cells
commonly, a boost is performed 3 days to keep the maximum of cells in the spleen. This results in a spleen that contains from one hundred to two hundred million spleen cells, with a majority of B cells capable of fusing and especially to secrete Ig.
I hope this will help you
Gualberto, did you experience failure with the same antigen as used successfully before, or antigen is distinct?
This is very unusual. First of all I would check the titer of antibodies in the serum of the animal, before fusion. Some antigens are not very immunogenic. Next I would check that the fusion worked, e.g. you got an expected number of hybridomas in your wells. If the antigen is immunogenic, of the fusion worked and if the selection in HAT medium worked, there is no reason why you should not get at least a few (5-10) positive wells. To keep those positives requires timely cloning and clones stability (the chromosome producing the reguired Ig is not lost). If your antigen is not immunogenic in mice or rats, try a different part of the protein as immunogen, or try to make a polyclonal in another host (guinea pig, chicken).
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