2 questions: myeloma cell lines and mice MABs - (Nov/21/2010 )
Dear all,
I wanted to post a couple of unrelated questions. I hope someone with more experience can answer them.
1) Is there any advantage/disadvantage in using either NSO or NS-1 myeloma cells for making hybridomas? I have read that NS-1 cells actually produce a non-secreted version of a antibody light chain. However, many labs seem to be using NS-1 for fusion. Are these cells really harmless for monoclonal antibodies? A second issue associated with this one, some companies offer myelomas that can grow in serum-free medium. Is there any disadvantage in choosing this kind of cells (for instance, are the Ab yields lower?)?
2) I have heard that it is possible to develop a mouse monoclonal antibody against a mouse protein. How is that possible? The protein should be recognized as a self-antigen and consequently, no immune response should develop, right? Is it still possible to do so with peptides?
I will appreciate your help a lot!
Hi there,
I am not exactly sure what the difference between NS0 and NS1 is, but we use NS1 and it works perfectly. It produces a truncated heavy chain mRNA, which is not translated, so the hybridomas secrete only one type of heavy chain. The only problem arises if you want to sequence the coding mRNA, in which case you have two. You can grow any hybridomas in serum free medium. The advantage is that you don't have bovine (or other type of serum) derived antibodies. Although the hybridomas tend to multiply a bit slower and stick together to provide each other growth factors.
Yes, you can immunize mice with a mouse protein and get antibodies. The reason for this is that self vs. non-self recognition is not 100% selective and depends on the context in which the protein is presented to the immune system. Although we have done a lot of immunizations using peptides I myself am not a fan of that, because the peptide never assumes the right conformation as is found in the native protein. I much prefer immunizing with whole proteins.
BioMiha on Mon Nov 22 05:45:51 2010 said:
Hi there,
I am not exactly sure what the difference between NS0 and NS1 is, but we use NS1 and it works perfectly. It produces a truncated heavy chain mRNA, which is not translated, so the hybridomas secrete only one type of heavy chain. The only problem arises if you want to sequence the coding mRNA, in which case you have two. You can grow any hybridomas in serum free medium. The advantage is that you don't have bovine (or other type of serum) derived antibodies. Although the hybridomas tend to multiply a bit slower and stick together to provide each other growth factors.
Yes, you can immunize mice with a mouse protein and get antibodies. The reason for this is that self vs. non-self recognition is not 100% selective and depends on the context in which the protein is presented to the immune system. Although we have done a lot of immunizations using peptides I myself am not a fan of that, because the peptide never assumes the right conformation as is found in the native protein. I much prefer immunizing with whole proteins.
Hi BioMiha,
thank you a lot for your prompt answer to my post. Could you advise me on how to grow the hybridomas in serum-free medium? My cells eventually die if I remove the serum (by gradually lowing the percentage). Thanks a lot in advance!
Basically what we do is, we thaw the cells directly in serum free medium. After that the viability drops dramatically (1. because of thawing of cells and 2. because of the sub-optimal medium). However if you remove the dead cells and replace fresh medium daily they start to grow. We observe that they like to grow in bunches, probably providing citokines and growth factors for one another. You have to break them apart by pushing the medium through a pipette to cause shear, otherwise the cells in the center of the bunch die and cause the ones on the outside to die as well. Although I have to be clear that if the frozen vial of cells is less than perfect meaning that these cells are not 100% viable, you can indeed kill them all by thawing them in serum free medium. Also, your serum free medium might lack certain components e.g. glutamate or other amino acids, so be sure to check if it is appropriate for your cells.
BioMiha on Thu Nov 25 13:54:41 2010 said:
Basically what we do is, we thaw the cells directly in serum free medium. After that the viability drops dramatically (1. because of thawing of cells and 2. because of the sub-optimal medium). However if you remove the dead cells and replace fresh medium daily they start to grow. We observe that they like to grow in bunches, probably providing citokines and growth factors for one another. You have to break them apart by pushing the medium through a pipette to cause shear, otherwise the cells in the center of the bunch die and cause the ones on the outside to die as well. Although I have to be clear that if the frozen vial of cells is less than perfect meaning that these cells are not 100% viable, you can indeed kill them all by thawing them in serum free medium. Also, your serum free medium might lack certain components e.g. glutamate or other amino acids, so be sure to check if it is appropriate for your cells.
Thanks a lot again, BioMiha! I will try your way. Apart from growth issues, is the antibody synthesis still ok?
Yes, once we get the cells to grow properly the Ab productivity is very high.