non IgG secreting hybridomas - (Feb/06/2006 )

Hi,

I'm producing mouse hybridomas. I have high titer of IgG in sera (by ELISA technique). After cell fusion, clones of hybridomas are produced but they did not secrete IgG, they secrete IgM. Could you help me? What could be the main problem?

thanks

Hi
Titer - (IgG) even to antigen won't always correspond to Mabs - in a good # of fusions IgM versus IgG secreting clones ( not specific for antigen) are in the 40%/40% ratio - with the other 20% being non-secreters. This is a rough # and I have seen some antigens give high titer and will make very few IgG producing clones (let alone antigen specific).

Some antigens are just weird - especially ones that are related to the immunological system.

Is an antibody to your antigen available commercially? If not it could have been troublesome for other folks too. Try immunizing rats versus mice - your species homology may be less there and you may have better results ( this has been dramaticaly the case for me with a couple antigens) - rat hybridomas are a bit trickier to stabilze and purify but by no means impossible.

Good luck

Thanks for your attention

Do you know if mycoplasma contamination could affect antibody secretion and isotype? We had a mycoplasma contamination two months before with other cells and I believe that my hybridomas could be affected.

Thanks for your attention

Do you know if mycoplasma contamination could affect antibody secretion and isotype? We had a mycoplasma contamination two months before with other cells and I believe that my hybridomas could be affected.

hi toxin if you are interested in clones producing IgG antibodies then you could force them for class switching of isotype by using CD40 specific ligand. how ever this would be a hit and trial method as you may even get clones producing IgA, IgE antibodies . you may have to standardise many things like amt. of CD40, time of stimulation, other factors required. take this just as a hint as i donot know the protocol exactly.
all the best

you can tell me your immunization project ,IgM normal produce in immunization early phage

hi toxin37

normaly say mycoplasma can affect antibody secretion,but it cannot affect isotype

hi,

in this discussion u can find good information, if u go through it, u may understand even better about monoclonal antibody production as we discussed about the serected antibody types at different time intervals.
http://www.protocol-online.org/forums/index.php?showtopic=3093

gud luk