I have obtained monoclonal cells of hybridoma by cell fusion assay, it grows well in hybridoma medium which is GIBCO brand and supplemented with 20% fetal bovine serum, L-glutamine, how can I purify the monoclonal antibody from the cell culture supernatant? Thanks！！！
Protein A or Protein G, immobilized on a carrier, commonly are used for this. Some companies are selling ready to use spin columns and columns for chromatograph.
The IgG from bovine serum however will cause you lots of trouble, as it co-purifies. Try to minimize the serum content (grow until confluence, then replace with serum free or low serum media) or better use appropriate media where specific growth factors have been added. Alternatively, you could use affinity chromatography on immobilized anti-mouse (your isotype) antibodies.
Alternatively, if you have the antigen, use affinity chromatography on immobilized antigen.
I prefer Ammonium sulfate precipitation, dialysis and then Protein A/G column purification. As Wolfgang Schechinger suggested, affinity chromatography with the antigen, if you have will work.
Hi Wolfgang,
when the concentration of your monoclonal is high enough, also the precipitation at the isoelectric point (by dialyzing vs. low molar buffer: 1 - 5 mmol/L) can be helpful. Beware of fragments of the antibodies: there are different (despite of the normal: H2L2): H2, L2, H1L1, H2L1 ...
Best
Stephan
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