I have the job of growing up two lines of IgM secreting hybridomas that have been grown up and frozen back down several times over the past 20 years (so they probably aren't in the best of shape anyways). One line just produces very little antibody and attempts at subcloning/sublining have yielded very subtle improvements in production, while the second line produces a good amount of IgM, but the antibody does not stain (in IHC) like older batches have (I've tried many dilutions of supernatant and NH4SO4 precipitate). Is it possible that the IgM that is secreted in the second line is no longer specific for the original target? Does anyone else have experience in trying to revive old hybridomas? I'm not sure how else to proceed besides more and more sublining to hopefully identify, by chance, viable and productive lines. Any input is appreciated. Thanks - Paul
The Ig genes have been rearranged so no, your hybrids as are not expressing a different Ig. Biologically speaking. Now, if your hybrids as are that old, I imagine that many users have use them will several thawing freezing circles and sometimes inappropriate handling. My first suggestion is to try to get one of the original vials (or as old as possible) from your nitrogen tank. Bring it up in 20% medium for your initial first week at least. Compare the efficacy of those antibodies with the ones you mentioned in your text. If you no longer have an old vial try to sequence your present hybrids as determine if some sort of mutation has happened during cell cycles. It may happen.
best of luck
It is common, that unstable successive cloning hybridomas need to stabilize the secretion of monoclonal antibody. It is common, some hybridomas are unstable, successive cloning need to stabilize the cell line and therefore secretion of monoclonal antibody. I agree with Bruno, you get the oldest cryotube you can, do you ensure cloning and producing the antibody recognizes its target.
Regards
Unfortunately Hybridoma lines are not noted for their robust antibody production. Since these lines are 20 years old, their handling over the years may be an issue. Antonio and Bruno give good advice to seek out the oldest, well stored, vial you can find and sub-clone from that to produce new stock tubes. The fact that one hybridoma seems to produce IgM, as it should, but the specificity is lost indicates that perhaps the cloning of this hybridoma was not done well. Over the years perhaps a non-specfic clone has taken over the culture. Again finding the oldest working vial may allow you to sub-clone a working cell out of the mix.
Is it possible to clone the IgM genes and then stabily transfect them into a high producing cell line?
Hi Paul,
you can try to reclone the V-genes in a mammalian expression vector.
The method is described in this paper: Frenzel, A. and Toleikis, L. (2012). Cloning single-chain antibody fragments (scFv) from hybridoma cells. Antibody Engineering: Methods and Protocols, Ed: Chames, P., Methods Molecular Biology 907:59-71
But keep following problems in mind:
- Hybridomas express often more than two antibody chains.
- The recombinant production of an IgM is a challenge
- The conversion of an IgM to scFv, Fab or IgG may result in an antibody which is not binding (a lot of IgM have very low affinities and binding to repeating epitopes > resulting in an avidity effect)
Michael
I agree with all the others, who answered before me. I think that it is imperative to go back to the oldest vial and to clone just after thawing, using a feeder layer (adherent macrophages from the peritoneum) and seeding different cells/well (5, 1.5, 0.5/microwell) so that you should have better change to get clones from the beginning.
Good luck
Unfortunately, I have already been working with the oldest cells that we have. I think that I will seed a new vial and start to subclone ASAP. As far as feeder cells go, I have mostly read about using spleenocytes, but are adherent macrophages a better choice? Thanks for all the input from everyone - Paul
yes you can also use macrophages by seeding 5000-6000 cells/well in 96 well plate, they are better choice for me because fresh macrophages are mostly out of risk of contamination specially mycoplasma and are equally good to speenocytes as feeder cells
If you are sure that you got by re-cloning an antibody producing cell line again, you should have an eye on the stability of the IgM. Most of those hybridoma producing IgM in different forms. They often loses their stabilization capacity by missing the J-chain so that you can find (by western blotting of the cell culture supernatant for heavy chains and light chains) IgM monomer, dimers … up to hexamers or even an IgM consisting out of one heavy chain and ono light chain only or even heavy chains only or L-chain dimers. You should check this at an early stage or re-cloning and select the subclones for complete IgM.
If the Light chain is lost, you would not find any antigen binding. Most of this other IgM variants can bind the antigen but very often not as the pentamer.
Hi Paul,
So if subcloning doesn't work out for you take a look at our hybridoma sequencing and recombinant expression service. Often (mutated) transcripts are still being expressed even though antibody expression and secretion have stopped. As a result you an still sequence the hybridoma. We have got quite a bit of experience in "fixing" sequences, so maybe we can help. Get in touch if you have any questions.
All the Best,
Mike
http://absoluteantibody.com/custom-services/hybridoma-sequencing/
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