Hybridoma culture not secreting mouse antibody - (Feb/15/2008 )
Hi everyone,
I've been growing an hybridoma secreting a mouse monoclonal antibody. Cells grow very well in RPMI supplemented with 10% FBS, L-glutamin, pen/strep and beta-mercaptoethanol. 
The color of my RPMI turn yellow rather quickly tough after a few days in culure.
The problem is when I purify the antibody on a protein A/G column (Pierce kit), I get about 1mg for 100ml of hybridoma supernatant. When I test the antibody in FACS analysis, I get no signal ! My test is done with a positive cell line for the tested Ag, and a previous Ab batch works just fine in parallel ! 
I tried again, taking supernatant before purification to do a FACS experiment. I got a low to medium positive signal, which I tought was due to low Ab concentration prior purification. But I got the same result again anyway.
Any idea of where the bug might happen ?
I've been growing an hybridoma secreting a mouse monoclonal antibody. Cells grow very well in RPMI supplemented with 10% FBS, L-glutamin, pen/strep and beta-mercaptoethanol.
The color of my RPMI turn yellow rather quickly tough after a few days in culure. The problem is when I purify the antibody on a protein A/G column (Pierce kit), I get about 1mg for 100ml of hybridoma supernatant. When I test the antibody in FACS analysis, I get no signal ! My test is done with a positive cell line for the tested Ag, and a previous Ab batch works just fine in parallel !
I tried again, taking supernatant before purification to do a FACS experiment. I got a low to medium positive signal, which I tought was due to low Ab concentration prior purification. But I got the same result again anyway.
Any idea of where the bug might happen ?
I think you may be better to use protein A column, instead of ProteinA/G column, because FBS antibodies bind strongly to protein G and do not bind (or very weakly bind) to protein A column .
Therefore the anibody purified (1mg) is mainly consisted of FBS antibodies with some of your desired mouse monoclonal antibody.
I think most of your mouse monoclonal antibody may be in the flowthrough...
Hope this may help.
During purification, you can use a volume of each fraction to dilute and make a direct ELISA to view the presence of IgG at the peak of binding and/or elution. You must detect IgG only coinciding with the peak of elution. If you can see IgG in the binding peak, you are losing antibodies along the purification.
I hope you serve
I would recommend you to use a serum free medium if you want to purify the antibodies with protein A/G columns
Following Minnie Mouse and Missele's thoughts I think you are purifying IgG in the FBS. We had that problem in my lab a while back, thought we had buckets of antibody, and it was indeed IgG from FCS, we then changed to use low IgG serum. We use to grow the cells in bags, so usually grow hybridomas in normal RPMI + 10%FCS to expand, and change to media with Low IgG FCS just before tranferring to bags (as this FCS is quite pricey), wash the cells several times in RPMI with no serum at all to remove traces of High IgG FCS, and the transfer to the low IgG media and grow for 1-2 weeks in the bags.
Good luck!
You may culture the hybridoma in serum free medium, but it is more expensive than normal medium that contains FCS.
Do you have to purify the Ab? Can't you use the supernatant directly? We do that a lot with hybridoma.