We have an IgM secreting hybridoma and want to convert it to a IgG. Any suggestions? Thanks a lot.
See paper by Spira G et al. in J Immun Methods 74:307, 1984
Personally I would clone the variable domains, put them on a vector that has the desired IgG isotype constant domains (H and L), and transient transfect to make IgG, or generate stable transfectants, in CHO or HEK293
You can digest the IgM pentamer into five individual IgG molecules, then purify them by HPLC or some other method.
Digesting IgM will not produce IgG molecules, sorry, they are different isotypes produced by different genes.
Correct, what I meant was that after digestion one will have 5 immunoglobulin molecules. Yes IgG1, IgA, IgE ,IgA and their subclasses are indeed different isotypes.
It is not possible to convert an IgM into an IgG. You must go back to the drawing board and find an IgG from your plated out hybridoma clones. We had a situation where we could only find IgM molecules, and never an IgG. This was repeated in another laboratory.
If the antibody has acceptable performance then why not work around it. We have several IgM Mabs which perform well in both Elisa and western blotting and there are good second antibody choices .
Good luck
We hope that the IgM has some therapeutic potential in oncology. We know this IgM can kill several breast tumor cell lines, but not others. However, so far very few clinical trials (3-4) have been involved in IgM. That is why we want a quick method to convert the IgM to IgGs.
I agree with Vahe Bedian, You will need to clone out the V domains genes from your IgM construct and put them into an IgG expression vector, chosing the IgG isobtype that you want.
Our hybridoma is making human IgM. We did make IgG1 based on the IgM CDRs, though the IgG1 appears to be ineffective in terms of cell binding and CDC.
IgM generally has low affnity. the IgG version harboring the the same VH/VL may lose activity due to the low avidity. you should either conduct affinity maturation or construct multi-valent antibody to improve the avididty. However recombinant multi-valent antibody tends to aggregate during the purification process. in my oppinion, you'd better re-make hybridoma to get a high affinity IgG vesion.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies