antibody purification - how to get rid of bovine IgG? (Jul/27/2007 )

Hi all,
i am purifying some monoclonal antibodies for activity assay from tissue culture supernatants using protein G column. The problem is that i am getting bovine IgG along with the monoclonal antibody which i am able to see as two distinct heavy chain bands on SDS-PAGE gel. also it seems that bovine IgG is more than the monoclonal antibody in the preparation. i want to assay the inhibition activity of the monoclonal antibody against a protein and this way i cannot quantify the minimal inhibitory concentration. someone suggested me to go about for ascites. but from literature i came to know that even there i would be getting other contaminant antibodies. so can someone please suggest me how to get pure monoclonal antibodies.
the protocol i am following is equilibrating the column with 20mM sodium phosphate buffer (pH 7.4), loading the supernatant after adjusting pH to 7.0 and elution with 100mM glycine pH 2.0
any suggestions are highly welcomed
thanking you all in advance
Leelaram

You can culture the hybridoma in special hybridoma culture medium (serum free), then you will not have any Bovine IgG in the monoclonal antibodies supernatant. In additon, the concentration of the antibody is higher in the serum free hybridoma culture medium than normal complete DMEM.

Hope this may help.

With ascites the same problem occur. Your Abs contaminated with mouse IgG.
Preparation of antibovine IgG affinity column is good variant but expensive one. For preparation of affinity column you should take near 10 mg per 1 ml resin. So it will be better to buy this column. It will be lower in cost in terms of your money and time Also you could apply IEC chromatography to isolate your mabs and purify from another Abs, because Abs from different species differentiate by their pI. To optimize conditions of IEC separation you should know pI of your mAbs and bovine Abs.
Also you could step decrease serum concentration to 0% in culture media, but it is not a case for each hybridoma cultures and dramatically decrease Mab production ( to 10ug\ml media).

For long term production of Mabs purified from other Abs I will advice you to see Fibercell system which allow you automatically get rid of bovine Abs ( because of hybridoma cells are cultured in compartment which separates from cell culture media by membrane with CuttOFF 50 kDa , so Abs from media will not contaminate your mAbs!