Hybridoma cells not secreting antibody - (Jan/25/2012 )

Dear colleagues,

This is my first time to deal with Hybridoma cell line. I am using the 11B11 clone which produce anti mouse IL4 monoclonal antibody. This cell line was purchased from ATCC. I am supposed it should be a genetically stable cell line? I've been growing the hybridoma cells for a while. Cells grow very well in RPMI supplemented with 10% FBS, L-glutamin, pen/strep . I split the cell line roughly every 4-5 days and collected the supernatant for antibody purification.

The problem is when I purify the antibody on a protein G column (millipore), I get about 5mg for 500ml of hybridoma supernatant. When I test the antibody by running the SDS gel. I got no bands for this antibody. Therefore, I also checked the supernatant before the purification. There is no signal as well. It told me that the cell line didn't secret the antibody from the beginning. The protein purified from hybridomas supernatant is just contaminated FBS.

I wonder if anyone has suggestions on how could I improve the production?


Thanks,
Latte

How did you check the original supernatant (dilutions, methods etc.)? Did you run some of your purified antibody on a gel to look for bands of the appropriate size for IgG?

I checked both the original supernatant and purified antibody by the SDS PAGE gel. I didn't see any expected bands.
For the original supernatant, I even concentrated the protein first before running the gel (it is roughly 10 times concentrated).
Therefore, I suspect the hybridoma cells didn't secret the antibody.

I should have read your post more carefully - sorry. I was interpreting what you had written to mean that you got no signal from a blot looking for the protein it is supposed to detect.

I don't think that the cell line needs to be induced to cause expression. It may pay to check out the original paper for the hybridoma, referenced on the ATCC website.